Bacillus Cellular and Molecular Biology

3rd Edition Peter Graumann

Visit to download the full and correct content document:
https://ebookmeta.com/product/bacillus-cellular-and-molecular-biology-3rd-edition-pet
er-graumann/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Cellular Molecular Mechanisms in Epigenetic

Evolutionary Biology

https://ebookmeta.com/product/cellular-molecular-mechanisms-in-
epigenetic-evolutionary-biology/

Cellular and Molecular Approaches in Fish Biology 1st

Edition

https://ebookmeta.com/product/cellular-and-molecular-approaches-
in-fish-biology-1st-edition/

Cellular and Molecular Immunology Abul K. Abbas

https://ebookmeta.com/product/cellular-and-molecular-immunology-
abul-k-abbas/

Cellular and Molecular Aspects of Myeloproliferative

Neoplasms - Part A (Volume 365) (International Review
of Cell and Molecular Biology, Volume 365) 1st Edition
Niccolo Bartalucci (Editor)
https://ebookmeta.com/product/cellular-and-molecular-aspects-of-
myeloproliferative-neoplasms-part-a-volume-365-international-
review-of-cell-and-molecular-biology-volume-365-1st-edition-
Oral Biology Molecular Techniques and Applications 3rd
Edition Gregory J. Seymour

https://ebookmeta.com/product/oral-biology-molecular-techniques-
and-applications-3rd-edition-gregory-j-seymour/

Cell Biology Genetics and Molecular Biology 2nd Edition

Dipak Kumar Kar

https://ebookmeta.com/product/cell-biology-genetics-and-
molecular-biology-2nd-edition-dipak-kumar-kar/

Ankylosing Spondylitis Axial Spondyloarthritis Cellular

Molecular and Environmental Factors Mohammad Hossein
Nicknam

https://ebookmeta.com/product/ankylosing-spondylitis-axial-
spondyloarthritis-cellular-molecular-and-environmental-factors-
mohammad-hossein-nicknam/

Cancer Principles and Practice of Oncology Primer of

the Molecular Biology of Cancer 3rd Edition Unknown

https://ebookmeta.com/product/cancer-principles-and-practice-of-
oncology-primer-of-the-molecular-biology-of-cancer-3rd-edition-
unknown/

Ankylosing Spondylitis Axial Spondyloarthritis Cellular

Molecular and Environmental Factors 1st Edition
Mohammad Hossein Nicknam

https://ebookmeta.com/product/ankylosing-spondylitis-axial-
spondyloarthritis-cellular-molecular-and-environmental-
factors-1st-edition-mohammad-hossein-nicknam/
 Bacillus
Cellular and Molecular Biology (Third edition)

Edited by
Peter L. Graumann

Caister Academic Press

Bacillus
Cellular and Molecular Biology (Third edition)

https://doi.org/10.21775/9781910190579

Edited by

Peter L. Graumann

Faculty of Chemistry and SYNMIKRO (Centre for Synthetic Microbiology)

University of Marburg
Marburg
Germany

Caister Academic Press

Copyright © 2017

Caister Academic Press

Norfolk, UK

www.caister.com

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

ISBN: 978-1-910190-57-9 (paperback)

ISBN: 978-1-910190-58-6 (ebook)

Description or mention of instrumentation, software, or other products in this book does

not imply endorsement by the author or publisher. The author and publisher do not assume
responsibility for the validity of any products or procedures mentioned or described in this book
or for the consequences of their use.

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system,
or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or
otherwise, without the prior permission of the publisher. No claim to original U.S. Government
works.

Cover design adapted from images provided by Felix Dempwolff (Indiana University,
Bloomington, IN, USA), images of different membrane proteins from Bacillus subtilis taken with
STED microscopy.

Ebooks
Ebooks supplied to individuals are single-user only and must not be reproduced, copied, stored
in a retrieval system, or distributed by any means, electronic, mechanical, photocopying, email,
internet or otherwise.

Ebooks supplied to academic libraries, corporations, government organizations, public libraries,

and school libraries are subject to the terms and conditions specified by the supplier.
 Contents

Prefacev

1 Replication of the Bacillus subtilis Chromosome1

Heath Murray, Tomas T. Richardson, Patrice Polard, Philippe Noirot and
Marie-Françoise Noirot-Gros

2 Dynamics of DNA Double-strand Break Repair in Bacillus subtilis35

Begoña Carrasco, Paula P. Cárdenas, Ester Serrano, Rubén Torres,
Elena M. Seco, Silvia Ayora and Juan C. Alonso

3 Chromosome Arrangement and Segregation 67

Peter L. Graumann

4 Cell Division 89
Frederico Gueiros-Filho

5 The Organization of Transcription and Translation 127

Peter Lewis and Xiao Yang

6 RNA-mediated Regulation in Bacillus subtilis155

Wade C. Winkler

7 General and Regulatory Proteolysis in Bacillus subtilis191

Kürşad Turgay

8 The Actin-like MreB ‘Cytoskeleton’ 223

Rut Carballido-López

9 Ins and Outs of the Bacillus subtilis Membrane Proteome 263

Jan Maarten van Dijl, Annette Dreisbach, Marcin J. Skwark, Mark J.J.B. Sibbald,
Harold Tjalsma, Jessica C. Zweers and Girbe Buist

10 The Cell Wall of Bacillus subtilis295

Danae Morales Angeles and Dirk-Jan Scheffers

11 Genomics and Cellular Biology of Endospore Formation 333

Patrick Eichenberger

12 Multicellularity and Social Behaviour in Bacillus subtilis367

José Eduardo González-Pastor
iv | Contents

13 Competence and Transformation 395

Berenike Maier

14 Swimming, Swarming and Sliding Motility in Bacillus subtilis415

Anna C. Hughes and Daniel B. Kearns

15 Nucleotide Second Messengers: (p)ppGpp and Cyclic Dinucleotides 439

Danny K. Fung, Brent W. Anderson, Jessica L. Tse and Jue D. Wang

Index467

Bacillus subtilis is one of the best understood
prokaryotes in terms of molecular biology and
cell biology. Its superb genetic amenability and
relatively large size have provided powerful tools
to investigate a bacterium in all possible aspects.
Recent improvements in fluorescence microscopy
techniques have provided novel and amazing insight
into the dynamic structure of a single-cell organism.
Research on B. subtilis has been at the forefront of
bacterial molecular biology and cytology, and
the organism is a model for differentiation, gene/
protein regulation and cell cycle events in bacteria.
The aim of this book is to present an overview of
the most recent exciting new research fields, and to
provide a picture of the major cytological aspects of
a bacterium, many of which are highly relevant for a
wide variety of bacteria.
Bacillus subtilis is a ubiquitous soil bacterium
that can be easily isolated from soil, using starch
as an energy source and relatively high salt con-
centration. Ideally, the soil sample is heated up
to 100°C for 30 min, allowing only for enduring
spores to be cultured from the sample. B. subtilis
is unique in that it can choose between at least
three different genetic programmes when nutri-
ents or other resources become scarce and/or cell
density reaches a critical threshold. To survive or
adapt to adverse condition, cells can either enter
stationary phase, which is characterized by the
formation of single motile cells (exponentially
growing cells contain a mixture of mostly non-
motile chains of cells and a few motile single cells),
can differentiate into enduring and metabolically
inactive spores, or, thirdly, can become competent
and take up DNA from the environment for acqui-
sition of new genetic material. In all three cases,
Preface
strikingly different genetic programs are turned
on that guide the cell through the differentiation
processes. In addition to this, B. subtilis shows
social behaviour, in that the cells communicate
with each other, and form multicellular structures
in the form of swarming cells and biofilms. Two
component systems, cascades of different sigma
factors, regulatory RNAs and specific proteolysis
of target proteins form an intricate regulatory net-
work, which is beginning to be unravelled, not only
in terms of specific modules, but also in terms of
whole complex processes that are connected with
each other. Specific mono- and di-nucleotides have
gained a lot of interest, as they regulate key steps in
bacterial growth and physiology. Most strikingly,
it has become clear that many proteins have spe-
cific subcellular addresses in bacterial cells. These
findings have established the field of ‘bacterial
cell biology’, and B. subtilis has been a forerunner
in this field. Many vital processes are disturbed
if proteins lose their specific localization, but the
fundamental question of how proteins are targeted
and specifically located in a call lacking intracellular
compartments is still unclear for most cases. There-
fore, it has become important to also study proteins
in terms of their localization within the cell, in addi-
tion to analysing their biochemistry and regulation.
This book is intended to show that we are beginning
to understand the way a bacterial cell functions as a
whole entity and in 3D, i.e. how it is spatially organ-
ized, and even how bacteria talk to each other, or
give their life for the sake of the whole community.
In this book, we will take and inside out approach
to look at Bacillus, starting with duplication of the
chromosome, cell cycle and transcriptional regula-
tion, following its MreB cytoskeleton underneath
vi | Preface
the cell membrane, through the membrane, to
the cell wall. Finally, we will consider the multi-
architectural processes of biofilm formation and
sporulation that embrace many of cytological and
genetical aspects throughout the cell. New added
chapters of the third edition cover the important
aspects of motility (Chapter 14) and regulation
through nucleotides (Chapter 15). As will become
apparent to the reader, many chapters overlap in
a variety of aspects, which is due to the fact that
most processes addressed in the book are intercon-
nected with each other. For example, the specific
localization of the replication machinery (Chapter
1) is an important aspect in DNA repair (Chap-
ter 2) and in ordered chromosome segregation
(Chapter 3), and also touches aspects of cell divi-
sion (Chapter 4). Amazingly, the actin-like MreB
cytoskeleton (covered in depth in Chapter 8) is
essential for viability, for the ordered insertion of
cell wall material (Chapter 10). The structure of
short and dynamic filaments appears to connect
and coordinate cell cycle events and rod-shaped
cell growth, showing that the cytoskeleton was
actually a functional prokaryotic invention. Many
processes thought to occur throughout the cell
or all over the membrane (Chapter 9) have been
found to be spatially confined to discrete regions,
which has shed light onto processes such as cell
division, replication, cell growth and sporulation.
Even though transcription and translation are
coupled in prokaryotes, these processes occur
at defined places within the cells (Chapter 5),
apparently facilitating ordered chromosome segre-
gation and efficient synthesis of highly expressed
proteins and of stable RNA. Regulation of tran-
scription through RNA molecules (Chapter 6) and
regulation of protein activity through proteolysis
(Chapter 7) have only recently been recognized as
major factors affecting bacterial physiology, and are
intertwined with the organization of transcription
(Chapter 5), sporulation (Chapter 11) and cell
division (Chapter 4). Like many bacteria. B. subtilis
cells form biofilms, which contain several distinct
subpopulations sharing different kinds of labour,
and which act rather as a multicellular organism.
This new concept in bacteriology is described in
Chapter 12. In the second edition, several impor-
tant recent findings in the rapidly moving fields
of research have been included, and the book has
had the addition of Chapter 13 dealing with the
ability of B. subtilis cells to take up DNA from the
environment and incorporate it into the chro-
mosome, when sufficient homology exists. This
developmental state is called ‘competence’ and is
described in detail. Importantly, the new chapter
also explains the molecular basis and mathematics
of the phenomenon called bistability, in which two
interchangeable B. subtilis subpopulations exist in
parallel that have distinct physiological states and
genetic programmes. This ability allows bacterial
populations to do ‘bet hedging’ and to be able
to respond to environmental conditions that may
(or may not) change in the near future. The third
edition has now captured important new develop-
ments from the recent 4 years, and has won two
important aspects in the life of a bacterium, motil-
ity (Chapter 14) and the regulation of cellular
signalling pathways via small nucleotides, of which
the important functions played by cyclic di-AMP
and cyclic di-GMP have only recently been recog-
nized, and are only slowly being understood at a
molecular level. Small nucleotides also affect the
lifestyle decision of bacteria whether to become
motile or stay sessile, and also motility is under
bistable control in B. subtilis, tying together several
chapters of this new edition.
Clearly, different fields in bacterial cell biol-
ogy and molecular biology are growing together,
providing a more and more integral view of the
bacterial cell.
My thank goes out to Gert Bange, who has
helped me in editing several chapters of this third
edition and to all authors, who have done a great
job updating the chapters.
Useful links
Several useful sites on the internet exist that
provide tools to study B. subtilis in more depth.
Foremost, the Bacillus sequencing consortium has
set up a site in which the whole genome of B. subti-
lis is accessible in a superb way. Subtiwiki is a new
site in which all genome data and gene expression
analyses can be obtained. Also, genotypes of many
strains with gene deletions are accessible via two
websites. Finally, most authors of this book have
websites for the interested reader to find further
information on the various research areas covered
in this book.
 Preface | vii

B. subtilis gene expression database W. Winkler, regulatory RNAs

http://subtiwiki.uni-goettingen.de/wiki/index.php/ http://www.wadewinkler.com
Main_Page
P. Lewis, organization of transcription
B. subtilis genome https://www.newcastle.edu.au/profile/peter-lewis
genolist.pasteur.fr/SubtiList/
K. Turgay, proteolysis
B. subtilis stock centre https://www.ifmb.uni-hannover.de/turgay.html
www.bgsc.org/
R. Carballido-Lopez, cytoskeleton
List of essential genes https://www.micalis.fr/Poles-et-Equipes/Pole-Biosys/
www.pnas.org/cgi/content/full/100/8/4678 ProCeD-Carballido-Lopez

H. Murray, replication J.M. van Dijl, membrane proteins

http://www.ncl.ac.uk/camb/staff/profile/heathmurray. w w w.r ug.nl/umc g/fac u lte it/d i sc ipl inegroepen/
html#background medischemicrobiologie/index

J.C. Alonso, DNA repair D.-J. Scheffers, cell wall

http://www.cnb.csic.es/index.php/en/research/research- http://www.rug.nl/staff/d.j.scheffers/
departments/microbial-biotechnology/genetic-stability
P. Eichenberger, sporulation
P.L. Graumann, DNA segregation http://biology.as.nyu.edu/object/PatrickEichenberger
htt p://s y nmi kro.com/de/for schung/zel lulaere-
organisation/peter-graumann.html E. Gonzales Pastor, biofilm formation https://cab.inta-csic.
es/en/investigadores/343/jose-eduardo-gonzalez-pastor
F. Gueiros Filho, cell division
http://www2.iq.usp.br/docente/?id=fgueiros
 Current Books of Interest
The CRISPR/Cas System: Emerging Technology and Application2017
Brewing Microbiology: Current Research, Omics and Microbial Ecology2017
Metagenomics: Current Advances and Emerging Concepts2017
Bacillus: Cellular and Molecular Biology (Third Edition)2017
Cyanobacteria: Omics and Manipulation2017
Foot-and-Mouth Disease Virus: Current Research and Emerging Trends2017
Brain-eating Amoebae: Biology and Pathogenesis of Naegleria fowleri2016
Staphylococcus: Genetics and Physiology2016
Chloroplasts: Current Research and Future Trends2016
Microbial Biodegradation: From Omics to Function and Application2016
Influenza: Current Research2016
MALDI-TOF Mass Spectrometry in Microbiology2016
Aspergillus and Penicillium in the Post-genomic Era2016
The Bacteriocins: Current Knowledge and Future Prospects2016
Omics in Plant Disease Resistance2016
Acidophiles: Life in Extremely Acidic Environments2016
Climate Change and Microbial Ecology: Current Research and Future Trends2016
Biofilms in Bioremediation: Current Research and Emerging Technologies2016
Microalgae: Current Research and Applications2016
Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and
New Perspectives2016
Virus Evolution: Current Research and Future Directions2016
Arboviruses: Molecular Biology, Evolution and Control2016
Shigella: Molecular and Cellular Biology2016
Aquatic Biofilms: Ecology, Water Quality and Wastewater Treatment2016
Alphaviruses: Current Biology2016
Thermophilic Microorganisms2015
Flow Cytometry in Microbiology: Technology and Applications2015
Probiotics and Prebiotics: Current Research and Future Trends2015
Epigenetics: Current Research and Emerging Trends2015
Corynebacterium glutamicum: From Systems Biology to Biotechnological
Applications2015
Advanced Vaccine Research Methods for the Decade of Vaccines2015
Antifungals: From Genomics to Resistance and the Development of Novel
Agents2015
Bacteria-Plant Interactions: Advanced Research and Future Trends2015
Aeromonas2015
Antibiotics: Current Innovations and Future Trends2015

Full details at www.caister.com

Replication of the Bacillus subtilis
Chromosome
Heath Murray1*, Tomas T. Richardson1, Patrice Polard2, Philippe
Noirot3 and Marie-Françoise Noirot-Gros3
1
Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle
Upon Tyne, UK.
2
Centre National de la Recherche Scientifique, Laboratoire de Microbiologie et Génétique Moléculaires, Université
de Toulouse, Toulouse, France.
3
Argonne National Laboratory, Argonne, IL, USA.
Correspondence: heath.murray@newcastle.ac.uk
https://doi.org/10.21775/9781910190579-01
Abstract
Eubacteria have evolved multicomponent protein
machines, termed replisomes, which duplicate
their chromosomes rapidly and accurately. Exten-
sive studies in the model bacteria Escherichia coli
and Bacillus subtilis have revealed that in addition
to the core replication machinery, other proteins
are necessary to form a functional replication fork.
Specific subsets of proteins mediate (a) assembly of
the replisome at the chromosomal origin of replica-
tion [initiation]; (b) progression of the replication
forks along the chromosome [elongation] and their
maintenance by providing solutions for replication
restart, which are adapted to overcome possible
‘roadblocks’ encountered on the DNA template; and
(c) physiological arrest of replication when chromo-
some duplication is completed [termination]. This
review summarizes recent knowledge about chro-
mosomal replication in Bacillus subtilis and related
Gram-positive bacteria. It is focused on the events
governing the assembly and fate of the replication
fork, describes protein networks connected with
the replisome, and emphasizes several novel aspects
of DNA replication in this group of bacteria.
Introduction
Bacillus subtilis duplicates its circular chromosome
by initiating DNA synthesis at a single locus, the
1
replication origin (oriC, 0°; see Fig. 3.1). Rep-
lication proceeds bidirectionally with the two
replication forks progressing in the clockwise and
counter-clockwise directions along the chromo-
some halves (Lemon et al., 2002). Chromosome
replication is completed when the forks reach
the terminus region (180°; see Fig. 3.1), which is
positioned opposite the origin on the chromosome
map and contains several short DNA sequences
(ter sites) that promote replication arrest (Duggin
and Wake, 2002). Specific proteins mediate all
of the steps in the DNA replication pathway. The
comparison between the sets of proteins involved
in chromosomal DNA replication in B. subtilis and
in E. coli reveals both similarities and differences
(Table 1.1). Although the basic components pro-
moting initiation, elongation, and termination of
replication are well conserved, some important
differences can be found (e.g. essential proteins
in one bacterium missing from the other). These
differences underline the diversity in the mecha-
nisms and strategies that various bacterial species
have adopted to carry out the duplication of their
genomes.
This review summarizes recent knowledge about
chromosomal replication in B. subtilis and related
Gram-positive bacteria. Detailed understanding
of the events governing the fate of a replication
fork in E. coli will be briefly described and used as
2 | Murray et al.

Table 1.1 Comparison of the B. subtilis and E. coli proteins involved in different aspects of chromosomal DNA
replication
B. subtilis E. coli Protein functiona References
DnaA DnaA Master initiator of DNA replication, oriC binding at Kornberg and Baker, 1992b;
DnaA-boxes and DnaA-trios, local unwinding of the DNA Krause et al., 1997; Moriya et al.,
duplex, and recruitment of DnaD 1990; Richardson et al., 2016
DnaB – Initiation at oriC, component of the replication restart Bruand et al., 1995; Bruand et
primosome, co-loader of the DnaC helicase al., 2001; Velten et al., 2003
DnaC DnaB Replicative helicase Velten et al., 2003
DnaD – Initiation at oriC, recruitment of DnaB, and component of Bruand et al., 2001; Bruand et
the replication restart primosome al., 2005; Rokop et al., 2004
DnaE DnaE b Class C DNA polymerase essential for replication fork Bruck et al., 2003; Dervyn et
progression, lagging strand polymerase, involved in al., 2001; Inoue et al., 2001; Le
error-prone synthesis and lesion bypass Chatelier et al., 2004; Sanders et
al., 2010
DnaG DnaG DNA primase, primer synthesis on lagging strand template Bird et al., 2000
DnaI – Co-loader of the DnaC helicase and component of the Bruand et al., 2001; Velten et al.,
replication restart primosome 2003
DnaN DnaN β-sliding clamp, processivity factor of the replicative Bruck and O’Donnell, 2000;
polymerases and binding hub for recruitment of alternative Ogasawara et al., 1986
polymerases and repair enzymes
– DnaC Loader of the replicative helicase Davey and O’Donnell, 2003
– DnaQ ε subunit of DNA polymerase III, proofreading (3′–5′) Scheuermann and Echols, 1984
exonuclease
– DnaT PriA-dependent replication restart primosome Sandler and Marians, 2000
DnaX DnaX τ subunit of DNA polymerase III, clamp loader, coupling of Lemon and Grossman, 1998;
leading and lagging strand synthesis McHenry, 2003
GyrA GyrA DNA gyrase subunit A, DNA breakage and rejoining Orr and Staudenbauer, 1982
GyrB GyrB DNA gyrase subunit B, ATP hydrolysis Orr and Staudenbauer, 1982
– Hda Regulatory inactivation of DnaA, hydrolysis of DnaA-bound Kato and Katayama, 2001;
ATP in the presence of sliding clamp loaded onto DNA Su’etsugu et al., 2004
HolA HolA δ subunit of DNA polymerase III, clamp loader wrench Bruck and O’Donnell, 2000;
Noirot-Gros et al., 2002
HolB HolB δ′ subunit of DNA polymerase III, clamp loader Bruck et al., 2005; Bruck and
O’Donnell, 2000
– HolC χ subunit of DNA polymerase III Johnson and O’Donnell, 2005
– HolD ψ subunit of DNA polymerase III Johnson and O’Donnell, 2005
– HolE θ subunit of DNA polymerase III, dispensable Johnson and O’Donnell, 2005
LigA Lig NAD-dependent ligase Petit and Ehrlich, 2000
PcrA UvrD, DNA helicase, essential for DNA replication and repair Petit et al., 1998; Petit and
Rep b Ehrlich, 2002
PolA PolA DNA polymerase I, removal of RNA primers by 5′–3′ Duigou et al., 2005; Yasuda and
exonuclolysis, gap filling and translesion DNA synthesis Okazaki, 1985
PolC – Replicative DNA polymerase with proofreading (3′–5′) Barnes et al., 1992; Bruck and
exonuclease activity O’Donnell, 2000; Sanjanwala and
Ganesan, 1991
PolY1 DinB b Y-family DNA polymerase, translesion synthesis in Duigou et al., 2004, 2005
association with PolA
PolY2 UmuC b SOS-induced Y-family DNA polymerase, translesion Duigou et al., 2004, 2005
synthesis in association with PolA, UV-induced
mutagenesis
 Construction and Maintenance of a Replication Fork | 3

Table 1.1 Continued

B. subtilis E. coli Protein functiona References
PriA PriA Replication restart primosome assembly, recognition of Marsin et al., 2001; Polard et al.,
forked DNA substrates, DNA helicase 2002
– PriB PriA-dependent replication restart primosome Sandler and Marians, 2000
– PriC PriC-dependent replication restart primosome, recognition Heller and Marians, 2005a;
of forked DNA substrates Sandler and Marians, 2000
– SeqA Regulation of initiation of chromosome replication by Lu et al., 1994
sequestration of hemimethylated GATC sites
SirA – Negative regulator of DnaA during sporulation, bind to Jameson et al., 2014; Rahn-Lee
domain I et al., 2009; Rahn-Lee et al.,
2011; Wagner et al., 2009
– Dam Methylation of GATC sites Lobner-Olesen et al., 2005
– DiaA Positive regulator of DnaA Ishida et al., 2004
– IciA Negative regulator of initiation of chromosome replication Thony et al., 1991
Soj – DnaA regulator, monomer inhibits DnaA filament formation, Murray and Errington, 2008;
dimer activates DnaA-dependent replication initiation Scholefield et al., 2012
SSB SSB Single-strand binding protein Polard et al., 2002
YabA – Regulation of DNA replication initiation, complex formation Noirot-Gros et al., 2002;
with DnaA and DnaN, inhibits DnaA filament formation Noirot-Gros et al., 2006;
Scholefield and Murray, 2013
YpcP – Potential 5′–3′ exonuclease, paralogous to the N-terminal Duigou et al., 2005
exonuclease domain of PolA, co-essential with PolA

aForproteins present in B. subtilis, function is summarized from studies performed in B. subtilis or related
Gram-positive bacteria. For proteins missing in B. subtilis, the function of the E. coli protein is indicated.
bThe E. coli protein is not the strict functional homologue of the B. subtilis protein.

a framework to compare and to highlight the dif-
ferences in molecular mechanisms. The process of
DNA replication will also be contextualized with
other cellular pathways.
The basics of DNA replication in
Escherichia coli
Replication fork assembly and
progression
The mechanics of DNA replication have been the
focus of several reviews (Langston et al., 2009;
McHenry, 2003, 2011; O’Donnell, 2006). The con-
struction of replication forks is a highly regulated
process mediated by protein-protein and pro-
tein–DNA interactions, through which a dynamic
molecular machine able to replicate both DNA
strands in a coordinated manner is assembled. The
E. coli replisome machinery comprises more than
a dozen proteins. Extensive biochemical, struc-
tural, and biophysical studies of the E. coli DNA
polymerase III holoenzyme (Pol III HE) have led
to a detailed molecular understanding of the dif-
ferent steps and activities that are necessary for its
assembly and for the progression of the replication
fork. Briefly, the E. coli Pol III HE consists of 10
subunits that assemble into a tripartite complex
composed of the catalytic DNA polymerase, the
β-clamp processivity factor DnaN, and the clamp
loader DnaX (with subunits τ/γ, δ, δ′, ψ and χ). The
DnaX complex assembles the dimeric β-clamp ring
so that it encircles the DNA template. Binding of
the β-clamp to the polymerase creates a tether that
holds the enzyme on the DNA. The DnaX complex
also coordinates the active DNA polymerases, thus
physically coupling the synthesis of the leading
(continuous) and lagging (discontinuous) strands.
Importantly, the leading strand DNA polymerase is
coupled to the helicase DnaB via a bridge formed
by the τ subunit. Helicase stimulates the efficiency
of DNA duplex unwinding and accelerates the rate
of fork progression. However, unwinding of the
DNA template by the replicative helicase gener-
ates topological constrains that must be removed
to allow complete duplication (and segregation) of
4 | Murray et al.
the newly replicated chromosomes; this is achieved
by the actions of DNA topoisomerases and DNA-
condensing proteins (see Chapter 3) (Hardy et
al., 2004; Schvartzman and Stasiak, 2004). The
DnaG primase interacts transiently with the DnaB
helicase and synthesizes RNA primers every 1–2 kb
for initiation of lagging-strand synthesis. The asso-
ciation of the helicase–primase complex with the
polymerase holoenzyme at the replication fork
completes the replisome. As the fork progresses,
ssDNA is generated on the lagging-strand template
and is coated with the single-stranded binding
protein (SSB) to inhibit secondary structures and
protect from nucleolytic attack. The RNA oligori-
bonucleotides deposited by primase are elongated
by DNA polymerase III up to the 5′ end of the next
RNA primer to form Okazaki fragments (Kornberg
and Baker, 1992b). Adjacent Okazaki fragments are
separated by a single-strand interruption (nick).
DNA polymerase I extends synthesis from the 3′
end of the nick and simultaneously degrades the
downstream RNA primer with its 5′–3′ exonucle-
ase activity. After removal of the RNA primer, the
nick is sealed by DNA ligase. The lagging strand
polymerase must rapidly dissociate from DNA
upon completing each Okazaki fragment, unbind-
ing from the used β-clamp before associating with
a new one. As the replication fork advances the
lagging strand polymerase is coupled to the leading
strand polymerase, resulting in the formation of a
DNA loop (‘trombone structure’) that repeatedly
grows and subsequently dissolves upon completion
of each Okazaki fragment. Advances in our under-
standing of the dynamics of DNA replication loops
at the fork have been reviewed (Geertsema and van
Oijen, 2013; Hamdan and van Oijen, 2010; Robin-
son and van Oijen, 2013).
Initiation of DNA replication at the
chromosome origin
Within the cell formation of an active replication
fork is a tightly controlled process. At the chro-
mosomal origin, oriC, initiation occurs only once
per cell cycle to ensure that the number of chro-
mosomes remains constant during exponential
growth (Boye et al., 2000). The replication origin,
oriC, is specifically bound and unwound by the
replication initiator protein DnaA (Kornberg and
Baker, 1992a; Messer, 2002; Mott and Berger,
2007). Bacteria have evolved diverse regulatory
mechanisms to control chromosomal replication
at the initiation step, which mainly regulate the
activity of DnaA and/or the accessibility of its
DNA sequence targets within oriC (Katayama et al.,
2010). Protein interactions between DnaA and the
DnaB helicase in complex with the DnaC protein
promote the loading of DnaB onto the unwound
region (Marszalek et al., 1996; Sutton et al., 1998).
The subsequent association of the DnaG primase
with the helicase results in the synthesis of an RNA
primer on the DNA template. The Pol III HE binds
to the primer end, and the DnaX–DnaB interaction
completes replication fork assembly.
Restart of blocked replication forks
As the replication fork progresses along the chro-
mosome it can encounter damage on the DNA
template. Some DNA damage can be skipped over
by the replisome, leaving the lesion behind in a
ssDNA gap for later recombinational repair (see
Chapter 2) (Langston and O’Donnell, 2006). How-
ever, some DNA damage sites have the potential to
stop replication and act as ‘roadblocks’ (McGlynn
and Lloyd, 2002; Michel et al., 2004). The persis-
tence of such blocks would prevent completion of
chromosome replication and consequently would
be lethal. Thus, in order to survive the cell must
activate damage-tolerance mechanisms, which
involve either bypass of the replicative block or its
repair, followed by the restart of replication.
Bypass of a replication block can be performed
by specialized low-fidelity DNA polymerases that
are capable of incorporating nucleotides opposite
damaged sites (Friedberg et al., 2005; Tippin et
al., 2004). Upon completion of this translesion
synthesis, which is often mutagenic (because an
incorrect base is inserted), the replicative poly-
merase resumes DNA synthesis with its usual high
fidelity. The shift from replicative to translesion
synthesis at sites of blocked forks involves the
polymerase sliding clamp (β2) being able to accom-
modate more than one polymerase at the same
time (Friedberg et al., 2005; Indiani et al., 2005).
The central role of the sliding clamp in modulating
DNA polymerase switching highlights the dynamic
nature of the replication machinery.
Alternatively, the E. coli replisome may stall
upon collision with a DNA binding protein such as
a co-transcribing RNA polymerase, but instead of
collapsing the replication fork remains intact and

later resumes synthesis (Pomerantz and O’Donnell,
2010). Single-molecule analysis has revealed the
capacity of the replicative DNA polymerase to hop
from one β clamp to another without leaving the
replication fork. Relatedly, it has been demonstrated
that mRNA transcripts can also be extended by the
leading strand polymerase, as occurs during lagging
strand replication (Pomerantz and O’Donnell,
2008). These observations support the notion that
DNA synthesis is likely to be discontinuous on
both strands.
In cases where the replisome encounters a
blocking DNA adduct or lesion that causes the fork
to disassemble, this deleterious event is overcome
by eliminating the cause of the arrest and then
directing the re-assembly of the replisome. Replica-
tion restart (RR) is mediated by specific proteins
that associate in various combinations to act on
different types of arrested forks (Marians, 2004;
Michel et al., 2004). For instance, recombination
intermediates can be used to generate forked DNA
onto which the replisome is reassembled (see also
Chapter 3). A key protein in this process is the PriA
helicase, which specifically binds forked-DNA and
ultimately promotes the recruitment of the DnaB
helicase and its loading onto ssDNA, followed by
the subsequent assembly of a functional replisome
(Kogoma, 1997; Michel et al., 2004; Sandler and
Marians, 2000; Xu and Marians, 2003).
Termination of replication
Replication forks end their progression along the E.
coli chromosome at physiological arrest sites in the
terminus region. The termination protein Tus binds
to ter sites on the DNA and the Tus-ter complex acts
by blocking the replication fork approaching from
one direction but not from the other (Neylon et al.,
2005). This way, a fork that has transversed the 180°
point will be arrested on the other chromosome
arm, until the slower fork arrives. The mechanism
that determines the polarity of the Tus-ter block
is a molecular mousetrap operating at the non-
permissive face of the Tus–ter complex (Berghuis
et al., 2015; Elshenawy et al., 2015; Mulcair et al.,
2006). The trap is set by the binding of Tus to the ter
site and is sprung by the incoming DnaB helicase,
which separates the strands within the Ter site and
triggers the formation of a locked Tus–ter complex
upon unwinding of a strictly conserved G-C base
pair. On the strand displaced by the DnaB helicase,
Construction and Maintenance of a Replication Fork | 5
the conserved cytosine residue in ter interacts with
a cryptic cytosine-specific site on Tus to form the
lock. The locked Tus–ter complex is extremely
stable and halts progression of the DnaB helicase.
However, the locked Tus-ter can be readily dissoci-
ated by a fork approaching from the permissive side
of the complex, thus allowing complete replication
of the terminus region (Mulcair et al., 2006).
Chromosomal DNA replication
in Bacillus subtilis
The DNA replication machinery
The replicases
B. subtilis DNA polymerase III, encoded by the
polC gene, is essential for chromosome replication
(Gass and Cozzarelli, 1973). The PolC polypep-
tide carries a DNA polymerase active site in its
C-terminal domain and a 3′–5′ proofreading exonu-
clease in its N-terminal domain (Barnes et al., 1992;
Sanjanwala and Ganesan, 1989, 1991). This is in
contrast to the E. coli DNA polymerase III in which
the polymerase and the proofreading exonuclease
activities are encoded by the dnaE (α subunit) and
dnaQ (ε subunit) genes, respectively (Table 1.1).
PolC also contains a zinc finger-like structure,
not present in the Gram-negative α subunit, that
tightly binds zinc and is essential for polymerase
function (Barnes et al., 1998). Genome sequence
analyses revealed that PolC is highly conserved in
low G+C Gram-positive bacteria (Lemon et al.,
2002). The PolC holoenzyme replicases from four
Gram-positive organisms, B. subtilis, Streptococcus
pyogenes, Staphylococcus aureus and Mycobacterium
tuberculosis have been reconstituted in vitro from
purified subunits (Bruck et al., 2005; Bruck and
O’Donnell, 2000; Gu et al., 2016; Sanders et al.,
2010). The polC gene (or dnaE1 and dnaQ genes for
M. t.), as well as the widely conserved dnaX, holA,
holB, dnaN and ssb genes, encoding the subunits τ,
δ, δ′, β and SSB, respectively, were expressed in E.
coli and the resulting proteins purified. The dnaX
genes from all Gram-positive species produce only
the full-length protein τ. This is in contrast to E. coli
dnaX, which encodes τ, the full-length product,
and γ, a shorter product generated by translational
frameshifting. Although the B. subtilis τ protein has
a similar domain organization to E. coli τ (Haroniti
6 | Murray et al.
et al., 2004; Martinez-Jimenez et al., 2002), atomic
force microscopy (AFM) revealed that it forms a
crescent-shaped structure that resembles the overall
configuration of the E. coli γ complex (Haroniti et
al., 2003, 2004;). The τδδ’ complex uses the energy
of ATP hydrolysis to open the β ring and load it as
a functional sliding clamp around the DNA. No
detectable homologues of the clamp loader ψ and
χ subunits, which increase the affinity of τ(γ) for δ
and δ′ in the DnaX complex of E. coli (McHenry,
2003), have been found in the genomes of Gram-
positive bacteria (Lemon et al., 2002). However, it
is possible that orthologous proteins could act to
stabilize the interactions within the clamp loader of
these organisms. In combination, the clamp loader
complex and the β clamp endow PolC with a high
speed (~700 nucleotides/second) and processiv-
ity that is comparable to that of the E. coli Pol III
holoenzyme. Thus, the reconstituted polymerase
complexes from B. subtilis, S. pyogenes, S. aureus,
and M. tuberculosis share the characteristics of
previously characterized Gram-negative replicases
(Bruck et al., 2005; Bruck and O’Donnell, 2000;
Gu et al., 2016). In addition, the pair wise interac-
tions between PolC, τ, δ, δ′ and β identified in these
reconstitution studies are also found between the
B. subtilis subunits (with the exception of the δ-δ′
interaction) using a yeast two-hybrid assay (Noirot-
Gros et al., 2002). This further underscores the
conserved organization of the PolC replicases in
Gram-positive bacteria.
A pentameric recognition sequence (consensus:
QL[S/D]LF) termed the clamp-binding motif
mediates the interaction of many proteins with
the β clamp (Dalrymple et al., 2001; Wijffels et al.,
2004) and is present at the C-terminus of virtually
all PolC proteins from low G+C Gram-positive
bacteria (Wijffels et al., 2005). Interestingly, the E.
coli α (polymerase) subunit contains two β-binding
motifs (Wijffels et al., 2005); one at the C-terminus
that is important for coordinating interactions
between β and τ (Lopez de Saro et al., 2003), and
one located internally that promotes β-binding in
vitro and is critical for replication in vivo (Dohr-
mann and McHenry, 2005).
In addition to PolC, many Gram-positive bacte-
ria also encode a second DNA polymerase, DnaE,
related to the E. coli Pol III α subunit. DnaE is essen-
tial for chromosome replication in B. subtilis and
S. aureus (Dervyn et al., 2001; Inoue et al., 2001).
Similarly to PolC, DnaE is required for the elonga-
tion phase of chromosome replication (Dervyn et
al., 2001). In DnaE-depleted cells, a plasmid with
unidirectional replication specifically produces a
single-stranded DNA corresponding to the leading
strand, indicating that DnaE is involved in lagging
strand synthesis. In addition, the subcellular locali-
zation of DnaE is similar to that of PolC, suggesting
that both polymerases are functioning at the B.
subtilis replication fork (Dervyn et al., 2001).
Recent work based on the in vitro reconstitu-
tion of an active B. subtilis replication fork brought
significant advances to the understanding of the
essential roles played by the two replicases during
DNA synthesis (Sanders et al., 2010). It was found
that B. subtilis PolC is the major DNA replicative
enzyme of the leading strand while both PolC and
DnaE are required for lagging-strand synthesis. In
the presence of the full complement of proteins that
compose the replisome machinery the rate of syn-
thesis by the PolC holoenzyme correlated well with
the progress of the replication fork in vivo (about
500 nt/s), while the reaction supported by DnaE
alone was slow (25 nt/s), even in the presence of τ
and β2. The authors also observed that both PolC
and DnaE could efficiently elongate a DNA primer,
but that under the conditions used only DnaE could
elongate an RNA primer. Finally, strong evidence
was provided for a handoff mechanism between
DnaE and PolC, whereby RNA primers are first
extended by DnaE and are then elongated by PolC
during lagging strand replication (Sanders et al.,
2010). Hence, the Bacillus replication machinery
assembled at active replication forks likely contains
two distinct replicative DNA polymerases, PolC
and DnaE. This architecture differs from that of E.
coli where identical PolIII enzymes are assembled
asymmetrically to efficiently replicate the two DNA
strands, but it is similar to the eukaryotic replisome
where a combination of Pol α and Pol δ are required
for lagging strand synthesis (Table 1.2). Thus, it
appears that the need for specialized leading and
lagging strand polymerases might be evolutionarily
conserved (McHenry, 2003).
The purified B. subtilis DnaE enzyme alone
lacks an associated proofreading 3′–5′ exonu-
clease activity but can synthesize DNA on an
SSB-coated ssDNA template at low speed (Bruck
and O’Donnell, 2000; Le Chatelier et al., 2004;
Sanders et al., 2010). The presence of the β sliding

Table 1.2 Differences in replication forks composition in E. coli, B. subtilis and yeast
Replisome factor E. coli
Leading strand polymerase Pol III
Lagging strand polymerase Pol III
Clamp β2
Clamp loader [τ/γ]δδ′ψ χ
Helicase DnaB
Helicase loader DnaC
Primase DnaG
SSB SSB
clamp increases the processivity of DnaE but not
its intrinsic speed (Bruck and O’Donnell, 2000).
Another striking property of the DnaE polymer-
ase is its capacity to bypass many types of DNA
lesions in vitro that generally block other replicative
polymerases (Bruck et al., 2003; Le Chatelier et al.,
2004). The bypass is efficient, highly error-prone,
and takes place either by extension of misaligned
3′ termini (thereby generating single base deletions
in the nascent strand) or by direct extension of
mispaired termini, depending on the nature of the
lesion (Bruck et al., 2003; Le Chatelier et al., 2004).
Notably, the expression of DnaE is induced about
3-fold upon treatment of B. subtilis cells with DNA-
damaging agents (Le Chatelier et al., 2004), and the
SOS repressor DinR binds to the dnaE promoter
region (Au et al., 2005). Although the expression
of DnaE is SOS-controlled (see Chapter 3 for
more details), DnaE is unlikely to function as an
error-prone polymerase in vivo because its overpro-
duction does not increase the rate of spontaneous
mutagenesis (as occurs following overproduction
of an error-prone Y-family polymerase; see below)
(Le Chatelier et al., 2004). A potential role of DnaE
in DNA repair and mutagenesis remains to be
investigated.
The primosome
The primosome is a complex between helicase and
primase that is required for primer synthesis during
DNA replication. Although primase alone can
catalyse oligoribonucleotide synthesis, this activ-
ity is increased 300-fold in the presence of helicase
(Tougu et al., 1994). The ordered assembly of the
primosome onto the DNA is directed at specific
sites, such as the chromosomal replication origin
Construction and Maintenance of a Replication Fork | 7
B. subtilis Eukaryotic
Pol C Pol δ, Pol ε
DnaE + Pol C Pol α + Pol δ
β2 PCNA
τδδ′ RFC
DnaC MCM2–7
DnaB/I (+ DnaD) Cdc6/Cdt1
DnaG Primase
SSB RPA
or at arrested forks, by the master initiator protein,
DnaA, and the helicase, PriA, respectively (see
above). In E. coli, the loading of the DnaB helicase
at oriC occurs through the formation of a DnaB6–
DnaC3 complex in which the N-terminal domain of
DnaC is physically associated with the C-terminal
domain of DnaB (Makowska-Grzyska and Kaguni,
2010) (Table 1.1). The transition from initiation
to elongation is triggered by the recruitment of the
primase, DnaG, to the N-terminus of DnaB and
concomitant dissociation of DnaC, to promote
the synthesis of RNA primers. This reaction of
‘general priming’ is inhibited in the presence of SSB
(Marians, 2000). In the absence of PriA E. coli cells
are deficient in the restart of arrested replication
forks and rapidly accumulate mutations in dnaC
that restore proficiency for restart. The DnaC810
suppressor protein has gained the capacity to
load DnaB onto SSB-coated DNA. These findings
indicate that in the cell, primosome assembly onto
ssDNA has to overcome SSB coating that acts as
a safeguard to prevent random initiation events at
single-stranded DNA regions in the chromosome
(Marians, 2000; Sandler and Marians, 2000).
In B. subtilis the dnaC and dnaG genes encode
the functional counterparts of the E. coli DnaB
helicase and DnaG primase, respectively, and the
initiator proteins DnaA and PriA are also conserved
(Table 1.1). However, the DnaD, DnaB and DnaI
proteins, which are components of the B. subtilis
primosome (Bruand et al., 1995; Bruand et al.,
2001), are not conserved in E. coli and conversely
the E. coli PriA partners, PriB, PriC and DnaT, are
not present in the B. subtilis genome (Lemon et al.,
2002). The loading of the E. coli replicative helicase
DnaB1 involves a homo-oligomeric DnaC complex
8 | Murray et al.
(Marians, 2000). In contrast, DnaD, DnaB, and
DnaI are all required to load the helicase, DnaC,1
at oriC or at stalled forks during replication restart
in B. subtilis.
The B. subtilis helicase loader, DnaB, is mul-
tifunctional; biochemical characterization has
shown that it cooperates with DnaI for the loading
of the helicase, DnaC, in vitro (Velten et al., 2003).
Evidence suggests that both DnaB and DnaD are
necessary to promote helicase loading at oriC and
that DnaB is recruited to the oriC–DnaA–DnaD
complex by interaction with DnaD (Rokop et al.,
2004; Smits et al., 2010). DnaB has been shown to
undergo proteolysis at its C-terminus in a growth
phase-dependent manner, which could play a role
regulating its interaction with DnaD and hence its
recruitment to oriC (Grainger et al., 2010). The
observation of structural similarities between DnaB
and DnaD is reflected in their ability to oligomerize
upon binding to DNA. Furthermore, DnaD pos-
sesses DNA remodelling activity on supercoiled
DNA, forming protein-DNA scaffolds that could
facilitate DnaA-dependent duplex unwinding at
oriC (Zhang et al., 2008).
B. subtilis DnaC belongs to the F4 family of
ATP-dependent annular hexameric helicases (Gor-
balenya, 1993). Purified DnaC self-assembles into
a homohexamer in the presence of ATP (Velten
et al., 2003). However, neither the DnaC mono-
mer nor the hexamer exhibits helicase activity in
vitro. This is in sharp contrast to the replicative
helicases of E. coli and Bacillus stearothermophilus
which alone display helicase activity in vitro (Bird
and Wigley, 1999; Davey and O’Donnell, 2003).
In B. subtilis, as well as in Geobacillus kaustophi-
lus, the ATPase DnaI interacts with DnaC in the
presence of ATP and promotes the formation of a
dodecameric complex containing 6 units of each
protein (Tsai et al., 2009; Velten et al., 2003).
The C-terminal domain of DnaI belongs to the
structurally well-characterized AAA+ superfamily
of ATPases (see Chapter 7), while the N-terminal
domain adopts a novel zinc-binding fold impor-
tant for interaction with DnaC (Loscha et al.,
2009). Interestingly, evidence suggests that DnaI
and DnaB act together to mediate the functional
1 It is important to note the nomenclature here; in E. coli
and B. stearothermophilus the replicative helicase is
DnaB, whereas in B. subtilis it is DnaC.
loading of DnaC onto ssDNA by promoting its
hexamerisation around the ssDNA (Velten et al.,
2003). The mutant protein DnaB75 (DnaBS371P),
which suppresses the defects of a priA null mutant
(Bruand et al., 2001), binds with higher affinity to
a forked DNA substrate and stimulates the DnaI-
dependent helicase activity of DnaC more than
wild-type DnaB (Velten et al., 2003). Together
with the observation that the conversion of circular
ssDNA into dsDNA is highly efficient in dnaB75
cells (Bruand et al., 2001), these findings are con-
sistent with the notion that the DnaB75 protein
has gained the capacity to promote helicase load-
ing onto SSB-coated ssDNA molecules (Velten et
al., 2003). Thus, as in E. coli, the coating of ssDNA
by SSB in wild-type B. subtilis cells may act as a
safeguard to prevent random initiation events, and
the capacity to deliver the replicative helicase onto
SSB-coated ssDNA likely is a general property of
helicase loading systems in bacteria.
The DnaG primase associates with the trans-
locating replicative helicase and deposits RNA
primers along the ssDNA template. This critical
interaction is mediated by the carboxy-terminal
domain of the primase (DnaG-Cter) both in E. coli
and in B. stearothermophilus2 (Soultanas, 2005).
The three-dimensional structures of the E. coli and
B. stearothermophilus DnaG-Cter domains reveal a
similar architecture composed of two subdomains;
a large helix bundle followed by a smaller helix
hairpin (Oakley et al., 2005; Syson et al., 2005).
The two subdomains have distinct functions;
the helix hairpin module alone is sufficient for
strong binding to the replicative helicase, but the
larger subdomain is required to stimulate helicase
ATPase activity. Remarkably, the DnaG-Cter
helix bundle is structurally homologous to the
N-terminal domain of E. coli DnaB helicase and
superposition of the domains reveals a network of
spatially conserved surface residues that are largely
invariant in the replicative helicases of 14 bacterial
species (Soultanas, 2005). The creation of chimeric
proteins in which the C-terminal region of DnaG
was swapped with the N-terminal region of DnaB
revealed that these domains are not only structrural
but also functional homologues (Chintakayala et
2 
Importantly, note that the nomenclature of the
replicative helicase is DnaB in E. coli and B.
stearothermophilus whereas it is DnaC in B. subtilis.

al., 2007). In B. stearothermophilus the DnaG pri-
mase interacts with the flexible linker that connects
the N- and C-terminal domains of the helicase,
inducing the hexameric helicase to adopt a 3-fold
symmetrical conformation (a trimer of dimers)
with three primase molecules bound to the helicase
ring (Soultanas, 2005; Thirlway et al., 2004). The
crystal structure of hexameric DnaB helicase com-
plexed with the helicase binding domain (HBD) of
DnaG revealed a distinct two-layered ring structure
formed by the two domains of DnaB (Bailey et al.,
2007).
Binding of B. stearothermophilus DnaG to the
replicative helicase reduces the length of synthe-
sized primers (Thirlway and Soultanas, 2006).
Some helicase mutants bind to DnaG but fail to
modulate the length of primers whereas other
helicase mutants inhibit primase activity. Taken
together, these observations indicate that within
the replisome helicase and primase are capable of
modulating each other’s activities (Chintakayala et
al., 2008; Soultanas, 2005; Thirlway and Soultanas,
2006).
The assembly of the replication machinery is
complete when the primosome connects with the
DNA polymerase holoenzyme via an interaction
between the τ subunit and helicase. The B. subtilis
τ protein interacts with the B. stearothermophilus
DnaB replicative helicase through its C-terminal
domain. This interaction stimulates helicase activ-
ity and is essential for rapid fork progression in E.
coli (see above). The architecture of the complex
revealed by atomic force microscopy suggests that
a crescent-shaped τ pentamer interacts with the
bell-shaped DnaB hexamer (Haroniti et al., 2003,
2004). The τ–helicase complex plays an additional
role by allosterically modulating primase activity
through a τ–DnaB−DnaG ternary complex (Chin-
takayala et al., 2009).
The role of SSB at the replicative fork
During fork progression, ssDNA corresponding
to the lagging strand template is generated by the
activity of the replicative helicase and subsequently
is coated by single-stranded DNA binding (SSB)
protein. SSBs protect exposed ssDNA and prevent
the formation of secondary structures at the fork
to promote replisome progression. SSB assembles
into a stable homotetramer and binds ssDNA
through its N-terminal OB (Oligonucleotide/
Construction and Maintenance of a Replication Fork | 9
Oligosaccharide-Binding) domain (Raghunathan
et al., 2000). Beyond its role in DNA binding, SSB
has been shown to physically interact with various
replication proteins which facilitates their recruit-
ment to forks. For instance, E. coli SSB interacts
with the χ subunit of the clamp loader complex and
with DnaG (Benkovic et al., 2001). These interac-
tions require a short evolutionarily conserved
amphipathic signature motif of about 10 residues
located at the tip of the SSB C-teminal domain
(Shereda et al., 2009).
In B. subtilis there are two SSB paralogues, SSB
(also called SsbA) and YwpH (SsbB), encoded
by the ssb and ywpH genes, respectively. The two
proteins share 63% identity in the N-terminal DNA
binding domain, but SsbB lacks the C-terminal
domain of SSB. SSB is essential for viability whereas
SsbB is dispensable (Kobayashi et al., 2003). Tran-
scription of ssb is high in fast growing cells and is
induced during the SOS response, whereas ywpH is
strongly induced during competence development
(Berka et al., 2002; Hahn et al., 2005; Lindner et
al., 2004). Thus, in B. subtilis, SSB displays a house-
keeping role during chromosome replication and
genome maintenance, whereas SsbB acts during
transformation, possibly by binding the incoming
ssDNA and facilitating its recombination into the
chromosome (see Chapter 13).
The role of B. subtilis SSB during progression
of the fork can be gauged by its binding partners.
Currently, the SSB C-terminus interaction network
is composed of 12 proteins involved in various
DNA metabolic pathways (including DNA repair),
all of which are enriched at the replicative fork via
the C-teminal domain of SSB (Costes et al., 2010).
Interestingly, it was found that DnaE-GFP did not
accumulate at the replisome in a strain expressing
an SSB derivative lacking its C-terminal interaction
motif. This suggests that DnaE accumutates at active
chromosomal forks through a physical interaction
with SSB whereas PolC is known to bind to other
components of the replisome machinary. Bacillus
SSB also binds the primary DNA replication restart
protein PriA and the DNA recombination effec-
tors RecG and RecQ (Lecointe et al., 2007). These
three DNA helicases were shown to be continually
associated with active replication forks. In the case
of PriA, repair activity at arrested forks was found to
require interaction with the SSB C-terminus bind-
ing motif (Lecointe et al., 2007). Thus, it appears
10 | Murray et al.
that SSB acts as a platform for the recruitment of
DNA-processing enzymes in anticipation of their
role in replication restart.
It has been found that both SSB and SsbB can
be phosphorylated on the conserved residue,
tyrosine-82, by the protein-tyrosine kinase, YwqD,
and dephosphorylated by the phosphatase, YwqE
(Mijakovic et al., 2006). Phosphorylation of SSB
appears to increase its affinity for ssDNA and is
reduced upon treatment of cells with the DNA
damaging agent, mitomycin C. Tyrosine phos-
phorylation of SSB also occurs in E. coli and in
Streptomyces coelicolor, indicating that it is an evolu-
tionarily conserved process. These findings suggest
that SSB phosphorylation may act to regulate some
as yet unknown aspects of DNA metabolism (Mija-
kovic et al., 2006).
Other replication proteins
In B. subtilis Okazaki fragments are processed by
DNA polymerase I, using its 5′–3′ exonuclease
activity to remove RNA primers and 5′–3′ poly-
merase activity to simultaneously fill the resulting
gaps (Okazaki et al., 1968; Tamanoi et al., 1977).
Nicks are subsequently sealed by a DNA ligase.
The B. subtilis chromosome encodes two ligases:
the NAD-dependent ligase, LigA (YerG), and the
ATP-dependent ligase, LigB (YkoU). LigA is essen-
tial for viability and fully complements the growth
and UV sensitivity defects of an E. coli ligts mutant
(Petit and Ehrlich, 2000), indicating that LigA acts
to seal Okazaki fragments processed by DNA Pol
I. LigB is not essential and participates in the non-
homologous end joining (NHEJ) pathway to repair
double-strand breaks (see Chapter 2) (Weller et al.,
2002).
In B. subtilis removal of the topological con-
straints generated as the replicative helicase
unwinds the DNA template is achieved by: the three
essential DNA topoisomerases Topoisomerase I
(TopA), DNA gyrase (GyrAB) and Topoisomerase
IV (ParCE); the general DNA binding protein
HBsu (Kohler and Marahiel, 1997); and the SMC-
ScpAB condensin complex which is required for
chromosome organization and segregation (see
Chapter 3) (Lindow et al., 2002; Mascarenhas et
al., 2002; Nolivos and Sherratt, 2014; Soppa et al.,
2002).
The DNA replication factory
The visualization of replisome proteins (e.g. PolC,
DnaE, τ, δ, δ′, β, SSB) in living cells, using functional
fusions with green fluorescent protein (GFP),
revealed localization predominantly at or near
midcell (Fig. 1.1) (Dervyn et al., 2001; Lemon and
Grossman, 1998; Meile et al., 2006). This midcell
localization of the replisome depends upon DNA
replication elongation and when replication is
blocked at a specific chromosomal locus this site
colocalizes with the DNA polymerase at midcell
(Lemon and Grossman, 1998, 2000). Upon release
of the block and resumption of DNA replication
the duplicated chromosome sites are segregated
towards opposite cell poles away from the centrally
located replisome. These data indicate that in B. sub-
tilis active DNA replication forks are predominantly
maintained near midcell, forming a replication fac-
tory.
The replication factory contains at least two
replisomes assembled at two forks, each replicating
one half of the chromosome (Lemon and Gross-
man, 1998). Although it most frequently appears in
discrete positions at midcell, time-lapse microscopy
has revealed that the factory is mobile around the
cell centre, suggesting that the factory is not rigidly
fixed at a specific midcell position (Migocki et al.,
2004). Also, a PolC-GFP focus can split into two
foci that will merge again later, suggesting that the
two replisomes in the factory are able to separate
and come back together during a given round of
replication (Migocki et al., 2004).
Interestingly, the master replication initiation
protein, DnaA, was found to be associated with the
replication machinery during most of the cell cycle
(Soufo et al., 2008). Likewise YabA and SirA, which
are negative regulators of DnaA, localize at the rep-
lication factory (Hayashi et al., 2005; Jameson et al.,
2014; Noirot-Gros et al., 2006) (Fig. 1.1). In addi-
tion DNA repair enzymes, such as MutS (mismatch
repair) and SbcC, are dynamically associated with
the factory during replication (Fig. 1.1) (Meile et
al., 2006; Simmons et al., 2008; Mascarenhas et al.,
2006). Taken together, these findings suggest that
many auxiliary proteins are recruited to the replica-
tion factory as and when required. These findings
also support the accumulating evidence that chro-
mosomal and extrachromosomal elements act at
 Construction and Maintenance of a Replication Fork | 11

Figure 1.1 Subcellular localization of proteins at the replication factory. (A) Left: Localization of key components
of the replication machinery: the replicative polymerase α-subunit, PolC; the τ-subunit, DnaX; the single strand
binding protein, SSB; and the C-family DNA polymerase DnaE. Green fluorescent protein (GFP)-fusions were
expressed in living cells, which were subsequently mounted on agarose slides and observed by fluorescent
microscopy. All four fusion proteins localize at the replication factory with typical single or dividing foci located at
or near the cell centre. Right: The localization of the β-clamp subunit, DnaN, and the Rad50-homologue, SbcC,
at the replication factory depends upon active DNA replication. Using a strain in which the expression of the
DnaA initiator is controlled by the IPTG-inducible promoter, Pspac, the formation of GFP-DnaN and GFP-SbcC
foci requires the presence of IPTG (Meile et al., 2006). (B) Left: Domain organization of DnaA and a crystal
structure showing the DnaA filament bound to ssDNA through the AAA+ domain (PDB: 3R8F). Right: DnaA
assembles into a filament specifically on a single DNA strand containing an array of DnaA-trios. DnaA filament
formation can be captured by cross-linking and visualized on SDS-PAGE as higher-order oligomeric species.
Assembly of the DnaA filament requires loading from double-stranded DnaA-boxes, is dependent upon ATP
and the ssDNA-binding residue Ile190, and occurs specifically on repeating trinucleotide sequences termed
DnaA-trios (3′-GAT-5′) (Richardson et al., 2016). (C) Living cells expressing various GFP-fusion proteins involved
in genome maintenance (indicated on the top of each set of panels) in ssb3+ cells (isogenic wild-type) or ssbΔ35
cells (possessing a truncation mutant of SSB lacking the C-terminal 35 amino acids). White arrowheads point
to visible GFP foci. The loss of distinct foci in the ssbΔ35 strain reveals that the SSB C-terminus is required
for localization of the DnaE (but not PolC) replicase at the fork, and for localization of SbcC and RecJ (but not
YabA) (Costes et al., 2010).
12 | Murray et al.
specific intracellular locations in bacteria (Bravo et
al., 2005).
Initiation of DNA replication at the
chromosomal origin
DnaA and oriC
The ubiquitous bacterial DNA replication initiator
DnaA is a multifunctional protein composed of
four distinct domains that act in concert to promote
opening of the DNA duplex and deposition of the
DNA replication machinery (Fig. 1.1B) (Mott and
Berger, 2007). The C-terminal domain IV contains
a helix–turn–helix motif that specifically recog-
nizes nine base-pair duplex DNA sequences called
‘DnaA-boxes’ (consensus 5′-TTATCCACA-3′).
Domain IV is connected to domain III by a flexible
α-helix, which is observed to adopt alternative con-
formations in different crystal structures of DnaA
proteins. Domain III is composed of an initiator
specific AAA+ motif, conserved amongst DNA
replication initiator complexes. The AAA+ motif
is capable of binding and hydrolysing ATP, and it
acts as the primary oligomerization determinant
for the protein. Additionally, domain III contains
the residues required for DnaA to interact with
single-stranded DNA. Domain II tethers domains
III/IV to domain I, and domain I acts as a protein
interaction hub that promotes DnaA dimerization
and facilitates loading of the replicative helicase.
A comparison of 104 different DnaA proteins has
shown that domains III and IV are the most similar
(Messer, 2002), indicating that the ATP-dependent
assembly of DnaA oligomers is likely to be a con-
served activity across bacterial species.
Oligomeric structures of DnaA in complex with
the non-hydrolysable ATP analog AMP-PCP have
been determined in the presence and absence of
single-stranded DNA (Duderstadt et al., 2011;
Erzberger et al., 2006). In both structures DnaA
assembles into a right-handed helical filament that
is built upon interactions between AAA+ motifs
and that requires a conserved ‘arginine finger’ to
contact the γ-phosphate of the nucleotide bound
by the adjacent protein. The structure of the
DnaAAMP-PCP:ssDNA complex revealed that each
DnaA protein contacts the phosphate backbone of
three nucleotides through the AAA+ motif. It has
been proposed that the DnaA filament stretches the
ssDNA to promote opening of the duplex replica-
tion origin.
Archetypical bacterial genomes have a unique
replication origin that contains several specific
binding sites for DnaA. The mechanism of DnaA-
mediated initiation at oriC has been historically
studied in E. coli (Messer, 2002), with significant
insights into DnaA structure coming from more
recent studies using the Aquifex aeolicus homo-
logue (Duderstadt et al., 2010, 2011; Erzberger et
al., 2002, 2006). DnaA first binds to high affinity
DnaA-boxes within oriC using its dsDNA bind-
ing motif in domain IV. Following ATP-binding,
DnaA assembles co-operatively onto lower affinity
sites, ultimately triggering DNA duplex unwinding
(Krause et al., 1997). Recently, a new replication
origin element called the DnaA-trio (3′-GAT-5′)
was identified within B. subtilis oriC (Richard-
son et al., 2016). The DnaA-trio is a repeating
trinucleotide motif that directs assembly of the
ATP-dependent DnaA filament onto one strand of
the DNA duplex promote open complex formation
(Fig. 1.1B).
DnaA-mediated unwinding of the origin is a
crucial step in initiation as it triggers loading of
the helicase onto ssDNA. In E. coli a direct inter-
action between DnaA and the helicase/DnaC
complex promotes the loading of helicase onto the
unwound DNA (Marszalek et al., 1996; Sutton et
al., 1998). In B. subtilis three initiation proteins,
DnaD, DnaB, and DnaI are required to assist
DnaA in the recruitment and loading of the repli-
cative helicase, DnaC, at oriC (Fig. 1.2) (Gross et
al., 1968; Imai et al., 2000; Karamata and Gross,
1970) (see above). DnaD directly interacts with
DnaA and DnaB (Bruand et al., 2005; Ishigo-Oka
et al., 2001; Rokop et al., 2004) and co-operates
with DnaB and DnaI to orchestrate the loading
of the replicative helicase, DnaC, around ssDNA
(Smits et al., 2010; Velten et al., 2003). It has been
found that these proteins are recruited to oriC in
a hierarchical order, with DnaD requiring DnaA,
DnaB requiring DnaA/DnaD, and helicase
requiring DnaA/DnaD/DnaB (Smits et al., 2010).
The assemblage of DnaD, DnaB and DnaI is called
the prepriming complex and, in contrast to E. coli,
these proteins are also components of the PriA-
dependent replication restart system (Bruand et
al., 1995, 2001).
 Construction and Maintenance of a Replication Fork | 13

Replication
A Initiation
Restart

oriC Arrested and repaired

fork

Lagging strand SSB

DnaA boxes

Leading strand

DnaA PriA
DnaD
(i) (i)

(ii)
DnaB DnaI

(iii)

DnaC Helicase

Helicase loading by
ring-assembly

Figure 1.2 The DnaA and PriA primosomes in B. subtilis. (A) The DnaA and PriA primosomes are two multiprotein
complexes that promote initiation of chromosome replication by the assembly of a pair of replisomes at oriC
(left), and replication restart by the assembly of a single replisome on arrested and repaired chromosomal forks
(right), respectively. DnaA and PriA specifically recognize loci at which primosome assembly is needed: DnaA
binds to an array of short dsDNA motifs (DnaA-boxes) within oriC; PriA targets forked DNA substrates with a
double-stranded leading-strand arm and a single-stranded lagging-strand arm. Once specifically bound to their
respective substrates, DnaA and PriA direct the recruitment and loading of the ring-shaped hexameric replicative
helicase, DnaC, around ssDNA at the tip of the fork. Importantly, DnaC is loaded on the lagging-strand template
where DNA replication is discontinuous. In subsequent steps (not shown) the loaded helicase recruits the
other components of the replisome. A hallmark of the B. subtilis DnaA and PriA primosomes is that they both
rely on the same triad of proteins to recruit and load DnaC, i.e. the essential DnaD, DnaB and DnaI proteins,
which are highly conserved in Gram-positive bacteria. Bi-directional arrows indicate the characterized physical
interactions between proteins, leading to the representation of two primosomal cascades initiated by DnaA and
PriA binding to their specific DNA substrates (unidirectional arrows). The cascades involve a series of protein
interactions: (i) first, DnaD is contacted either by DnaA or PriA bound to DNA; (ii) next, DnaB is contacted by
DnaD and helps overcome the SSB barrier; (iii) then, DnaC is dually contacted by DnaB and DnaI, which leads
to the functional loading of the helicase as a hexamer encircling the ssDNA. The loading takes place through
a ring-assembly mechanism, which relies on the recruitment of individual monomers of DnaC by DnaI and
DnaB on ssDNA to promote DnaC hexamerization around the ssDNA template. Thick arrowheads indicate the
direction of replication. (B) SSBCter interactome at the active fork. SSB tetramers act as a platform for recruiting
partners involved in fork restart upon adventitious blockage. The replisome machinery re-assembles on the
branched DNA backbone of the fork. The recruitment of PriA is crucial and takes place when arrest results from
replisome dismantling. In more complex situations other actions, aimed at protecting and/or clearing the fork,
may occur via individual or concerted actions of the many members of the SSBCter interactome.
14 | Murray et al.
The E. coli replication origin contains specific
binding sites for accessory proteins like IHF
and Fis that facilitate origin unwinding (Messer,
2002). In B. subtilis it is not known whether
sequence-specific DNA binding proteins other
than DnaA associate with oriC during initiation.
Potentially the DNA-remodelling activities of
DnaD and/or DnaB could play roles analogous
to IHF/Fis to facilitate open complex formation
(Carneiro et al., 2006; Turner et al., 2004; Zhang
et al., 2005).
The control of initiation
In bacteria, chromosome replication is tightly
regulated to ensure that initiation at the chromo-
somal origin takes place only once per cell cycle
(Boye et al., 2000). This regulation involves a
balance between triggering initiation at a specific
time of the cell cycle and repressing it to prevent
extra initiation events. Different initiation control
pathways have evolved to regulate the activity
and the availability of DnaA during the cell cycle
(Katayama, 2001; Marczynski and Shapiro, 2002).
In E. coli, the paradigm for initiation studies,
the predominant mechanism that prevents over-
initiation is the regulatory inactivation of DnaA,
termed RIDA, which acts after initiation to pro-
mote the switch from active ATP-DnaA to the
inactive ADP-DnaA form. RIDA is mediated by
Hda, a protein that is paralogous to DnaA and
whose activity requires interaction with the β
clamp (Camara et al., 2005; Katayama et al., 1998;
Kato and Katayama, 2001; Su’etsugu et al., 2005).
A second level of regulation is the sequestration
of newly replicated oriC regions by the SeqA pro-
tein (Lu et al., 1994; Taghbalout et al., 2000; von
Freiesleben et al., 1994). SeqA binds with high
affinity to hemimethylated dam sites (GATC)
found throughout the genome and especially
enriched at oriC and within the dnaA promoter. In
doing so, SeqA temporarily restrains re-initiation
both by preventing DnaA access to oriC and by
inhibiting DnaA expression (Campbell and Kleck-
ner, 1990). The third level of initiation control
involves the titration of large amounts of DnaA
protein to the datA locus (Kitagawa et al., 1998),
which stimulates hydrolysis of the DnaA-bound
ATP and lowers the concentration of active DnaA
in the cell (Kasho and Katayama, 2013; Ogawa et
al., 2002).
DiaA is a positive regulator of DnaA and is
required to ensure timely initiation of chromosome
replication (Ishida et al., 2004). The structure of
DiaA and functional analysis of defective mutants
revealed that it binds as a tetramer to multiple
DnaA molecules to stimulate inter-DnaA interac-
tions (Keyamura et al., 2007). DiaA is proposed to
positively regulate ATP–DnaA assembly, thus pro-
moting the formation of the open complex at oriC
(Keyamura et al., 2009). In addition, two different
mechanisms have been described that regenerate
ADP-DnaA into active ATP-DnaA. One relies on
an interaction of ADP-DnaA with acidic phospho-
lipids present in the membrane (Boeneman and
Crooke, 2005) and the other involves two spe-
cific chromosomal loci called DnaA-reactivating
sequences (DARS1 and DARS2) (Fujimitsu et
al., 2009). All together this network of control
systems contributes to proper regulation of DNA
replication in order to maintain a once-per-cell-
cycle initiation rate in E. coli cells. Although well
characterized, the conservation of these regulatory
proteins is limited to the Enterobacteriaceae and
other bacteria appear to have developed distinct
mechanisms for initiation control.
In B. subtilis there are no homologues of E. coli
Hda, SeqA, DiaA or Dam. Nonetheless, DNA
replication initiation is regulated as tightly as in E.
coli (Lemon et al., 2002; Moriya et al., 1999) and
several mutants affecting the regulation of ini-
tiation have been isolated. The B. subtilis initiation
regulator, YabA, was revealed in a yeast two-hybrid
screen to identify the protein interaction network
of the DNA replication machinery (Noirot-Gros
et al., 2002). A ΔyabA mutant exhibits over-
initiation and replication asynchrony phenotypes,
indicating that YabA acts as a negative regulator
of initiation (Noirot-Gros et al., 2002). YabA
assembles into a tetramer mediated through its
N-terminal domain and interacts with both DnaA
and the β clamp through its C-terminal domain
(Felicori et al., 2016; Noirot-Gros et al., 2006).
Although YabA and E. coli Hda share the capacity
to interact with both DnaA and DnaN, YabA is
distinct from Hda in that it has no homology with
DnaA and does not contain a consensus motif for
binding to DnaN (Kurz et al., 2004). This indi-
cates that YabA lacks the key structural elements
of Hda required to promote DnaA-ATP hydroly-
sis, as occurs in the RIDA system (Su’etsugu et al.,

2005). Thus, although YabA regulates initiation
through a novel mechanism, the involvement of
interactions with DnaA and DnaN suggests a con-
served coupling between initiation and elongation
of replication.
Live cell fluorescence microscopy has shown
that YabA localizes at the replication factory during
most of the cell cycle through its interaction with
DnaN (Hayashi et al., 2005; Noirot-Gros et al.,
2006). Moreover, YabA was shown to confine GFP-
DnaA at the replisome during most of the cell cycle
(Soufo et al., 2008), leading to the model that YabA
prevents untimely reinitiation by mediating spatial
sequestration of DnaA away from oriC (Noirot-
Gros et al., 2006; Soufo et al., 2008).
However, the sequestration model predicts
that YabA would titrate DnaA away from other
chromosomal loci known to bind DnaA, yet global
gene expression in a yabA null mutant revealed that
the expression of genes controlled by DnaA was
not altered (Goranov et al., 2009) and the absence
of YabA did not increase the binding efficiency of
DnaA at DnaA-boxes throughout the chromo-
some (Cho et al., 2008). These results suggest that
the effect of YabA on DnaA activity is somehow
localized to oriC. Mutagenesis indicates that YabA
interacts with the AAA+ oligomerizaton domain
of DnaA (Cho et al., 2008). Moreover, it has been
found that YabA inhibits DnaA filament formation,
which could explain this oriC-specific regulation
(Scholefield and Murray, 2013).
Soj is a dynamic Walker-type ATPase that is
directly regulated by the DNA-binding protein
Spo0J (Soj and Spo0J are members of the ParA
and ParB family of partition proteins). Soj oli-
gomerization state is regulated by ATP (Leonard
et al., 2005). Monomeric Soj (Soj-ADP) has been
found to physically interact with domain IIIb of
DnaA and to inhibit DNA replication initiation by
blocking DnaA filament formation (Murray and
Errington, 2008; Scholefield et al., 2012). In the
absence of Spo0J, or when expressed as a mutated
form deficient for ATP hydrolysis (SojD40A), Soj-
ATP dimerizes and DnaA-dependent initiation is
stimulated (Lee et al., 2003; Murray and Errington,
2008; Ogura et al., 2003). It has been shown that
Spo0J stimulates the ATPase activity of Soj, driv-
ing it from a dimer to a monomer and triggering
a switch from an activator to an inhibitor of DNA
replication initiation (Scholefield et al., 2011).
Construction and Maintenance of a Replication Fork | 15
However, it remains unknown when this switch is
activated during the B. subtilis cell cycle.
Genome-wide chromatin immunoprecipita-
tion assays have revealed that DnaA is significantly
enriched at six regions on the chromosome that
contain clusters of DnaA-box sequences (Breier
and Grossman, 2009; Ishikawa et al., 2007). A dele-
tion mutant lacking these six DnaA–box clusters
(DBC) overinitiated DNA replication (Okumura
et al., 2012), similar to the datA locus in E. coli.
Reintroduction of a single DBC at an ectopic posi-
tion near oriC, or reintroduction of multiple DBCs
at an ectopic position near terC, suppressed the
overinitiation phenotype, suggesting that a criti-
cal dosage of DBCs are required to regulate DnaA
activity. However, a multicopy plasmid harbouring
a DBC did not complement the mutant phenotype,
indicating that the DBC acts in cis. The intriguing
mechanism underlying DBC activity is yet to be
determined.
Replication restart after adventitious
arrest
In B. subtilis and in E. coli the genome is a large
circular molecule of over 4 million basepairs and
because of their single replication origin each
fork must travel more than 2 Mbp to achieve the
complete duplication of the chromosome. Conse-
quently, any accidental arrest suffered by one of the
forks represents a threat to cell cycle completion,
and thus cellular pathways have evolved to facilitate
the restart of arrested forks (Michel et al., 2004).
Intensive studies conducted in E. coli have uncov-
ered multiple responses to overcome the different
types of fork arrest that can be provoked by a vari-
ety of events (DNA damage, DNA-bound proteins,
and higher order DNA structures). These responses
range from simple pausing in the case of a protein
block, to induction of appropriate repair pathways
followed by re-activation of the fork and resump-
tion of chromosome replication. Replication fork
re-activation most often relies on the re-assembly
of the replisome on a processed arrested fork. This
event, termed ‘replication restart’ (RR), is directed
by a specific set of RR proteins (McGlynn and
Lloyd, 2002; Sandler and Marians, 2000).
The pathways of replication restart
In E. coli, the RR proteins PriA, PriB, PriC and
DnaT drive origin-independent initiation at sites
16 | Murray et al.
of collapsed replication forks (Table 1.1) (Marians,
2000). PriA plays a central role in replication fork
reactivation by triggering the serial assembly of the
downstream RR factors into a multiprotein com-
plex at the stalled fork. This RR prepriming complex
recruits the DnaB replicative helicase in association
with its loader DnaC to promote the subsequent
assembly of the replisome. There are three pathways
for restart in vivo based on the composition of the
prepriming complex: PriA–PriB–DnaT, PriA–PriC,
and PriC–Rep. The different complexes recognize
structurally distinct forked DNA molecules (Heller
and Marians, 2005a). The assembly of the repli-
some onto forked DNA molecules mediated by RR
proteins parallels the pathway mediated by DnaA
at oriC, where the replicative helicase is the first
partner to be recruited and loaded onto the lagging
strand template at the forefront of the replication
fork (Marians, 2000). However, whereas DnaA
binds specifically to repeated DNA motifs within
the origin, RR proteins recognize specific forked
DNA substrates. In the cell such structures origi-
nate from randomly arrested replication forks that
can be processed by various DNA repair pathways
and from DNA recombination intermediates (Fig.
2.2) (Michel et al., 2004). Thus, in contrast to DNA
replication initiation at oriC, the RR apparatus
has to deal with many different types of forked
DNA molecules. A key distinction between the
various forked DNA substrates is whether or not
the leading and lagging strand arms of the fork are
single-stranded. PriA is a DNA helicase belong-
ing to the large superfamily 2 (SF2) (Gorbalenya,
1993). PriA preferentially unwinds forked DNA
substrates with a double-stranded leading-strand
template to produce the ssDNA needed for delivery
of the replicative helicase, a fork remodelling activ-
ity that is crucial in the PriA and PriC pathways
(Heller and Marians, 2005b).
A common characteristic between the PriA-
dependent RR systems of E. coli and B. subtilis is that
PriA is not essential for growth. However, E. coli and
B. subtilis priA null mutant cells are poorly viable
and exhibit severe defects that can all be explained
by their failure to efficiently restart arrested forks.
Indeed, the defects of priA null mutants can be
almost fully suppressed by mutations that enhance
the loading apparatus of the replicative helicase,
DnaC in E. coli and DnaB in B. subtilis (Bruand et
al., 2001; Sandler et al., 1996). This indicates that
PriA controls the major RR pathway in wild-type
cells in both bacteria. In E. coli, the combination
of priA and priC null mutations is lethal, providing
strong evidence that RR is essential for cell survival
(Sandler, 2000). In B. subtilis, there is no PriC
homologue encoded in the genome, and only the
PriA-dependent RR pathway is well characterized.
However, genetic evidence supports the notion
that DnaD, which is a direct partner of PriA, pro-
motes RR in the absence of PriA (Bruand et al.,
2001). The presence of redundant RR pathways not
only provides a ‘backup’ system in case the initial
system fails, but also facilitates the targeting and
efficient processing of the distinct forked DNA
structures that can be produced upon replication
arrest (Heller and Marians, 2005a,b, 2006).
PriA-dependent replication restart
With the exception of PriA, the B. subtilis RR pro-
teins are distinct from those of E. coli (Table 1.1).
Notably, B. subtilis PriA and DnaA use the same
essential DnaD, DnaB and DnaI proteins to recruit
and load the DnaC replicative helicase onto ssDNA
(Fig. 1.2A) (Bruand and Ehrlich, 1995; Bruand
et al., 1995, 2001). B. subtilis PriA is functionally
similar to its E. coli counterpart, as it is a forked
DNA-binding helicase that acts as the primary RR
protein in vivo (Marsin et al., 2001; Masai et al.,
1999; Polard et al., 2002). However, B. subtilis PriA
cannot complement an E. coli priA mutant (Masai et
al., 1999; Polard et al., 2002). B. subtilis PriA initiates
a cascade of events starting with the recognition of
arrested forks in the chromosome, the sequential
recruitment of DnaD, DnaB, DnaI and finally
the functional loading of the DnaC helicase onto
ssDNA (Fig. 1.2) (Bruand et al., 2005; Marsin et
al., 2001; Velten et al., 2003). A remarkable feature
of the DnaD dimer and DnaB tetramer is that both
are non-specific DNA binding proteins (Marsin et
al., 2001; Zhang et al., 2005). Compelling evidence
indicates that the essential activities of DnaD and
DnaB during both chromosomal replication ini-
tiation and RR rely on their distinct DNA binding
activities at loci targeted by DnaA and PriA. The
association of DnaD and DnaB with chromosomal
regions bound by DnaA, including oriC, was con-
firmed by chromatin immunoprecipitation (ChIP)
(Smits et al., 2011). Furthermore, a genome-wide
analysis of DnaD and DnaB binding sites by ChIP-
chip indicated that the helicase loader proteins can

be associated with the highly transcribed regions of
rRNA (rrn) genes. The association of DnaD, DnaB,
and DnaC with rrn loci depends on transcription
and on the replication restart protein PriA (Mer-
rikh et al., 2011), strongly suggesting that DnaD
and DnaB are acting to reload the helicase for rep-
lication restart at forks stalled upon encountering
RNA polymerases.
DnaD and DnaB proteins have an architectural
role on DNA that facilitates the co-ordination, and
perhaps the licensing, of DnaI-dependent loading
of DnaC onto ssDNA (Bruand et al., 2005; Rokop
et al., 2004). The affinity of DnaB for ssDNA is
enhanced upon its interaction with DnaD (Marsin
et al., 2001) and the dnaB75 mutation suppresses
the thermosensitivity of the dnaD23 mutant
strain (Bruand et al., 2005; Rokop et al., 2004),
suggesting that these proteins act in combination
with one another. Consistent with this notion,
the DnaD23 protein displays a diminished and
thermosensitive ssDNA binding activity, whereas
the DnaB75 mutant protein exhibits a higher
affinity for ssDNA than the wild-type protein
(Bruand et al., 2005). Moreover, while neither
DnaD nor DnaB nor DnaB75 alone can interact
with ssDNA coated by SSB, which is known to
create a physical barrier to the loading of the
helicase in the E. coli system (see above) (Mar-
ians, 2000), together DnaD and DnaB proteins
can associate with SSB-coated ssDNA and this
binding is more efficient in the presence of the
DnaB75 derivative (Bruand et al., 2005). In addi-
tion, DnaB promotes DnaI-dependent helicase
loading and again this stimulation is more efficient
for the DnaB75 protein (Velten et al., 2003). Thus,
the concerted actions of DnaD and DnaB likely
provide a way to overcome the SSB barrier and
facilitate the subsequent DnaI-dependent loading
of the helicase on ssDNA (Bruand et al., 2005).
This effect could account for the observation that
the DnaD-, DnaB-, DnaI- and DnaC-dependent
replication of a ssDNA circular template produced
by a rolling circle plasmid is more effective in B.
subtilis cells expressing the mutant DnaB75 pro-
tein than the wild type DnaB protein (Bruand et
al., 2001). Finally, DnaD and DnaB can interact
in a distinctive manner with supercoiled DNA in
vitro, producing open and compacted DNA struc-
tures, respectively (Carneiro et al., 2006; Zhang
et al., 2005). These opposite DNA-remodelling
Construction and Maintenance of a Replication Fork | 17
activities of DnaD and DnaB could modulate
local chromosome architecture to facilitate the
DnaA- and PriA-mediated initiation events.
SSB as an organizer of the replication
fork maintenance complex
As stated above, SSB plays a pivotal role in the
recruitment of accessory proteins to active forks.
The E. coli SSB interactome is currently estimated
to comprise 14 proteins involved in various DNA
pathways. Parallels can be drawn with B. subtilis
where SSB is described as a protein-hub able to
target 12 known players at active replication forks
(Costes et al., 2010). In addition to PriA, RecG and
RecQ helicases, SSB also interacts with the RecQ-
homologue RecS in complex with YpbB and with
the putative helicase/nuclease, YrrC. The need for
PriA to interact with SSB in order to act efficiently
in fork restart has been demonstrated (Lecointe et
al., 2007). Similarly, a physical interaction of E. coli
RecG with SSB was shown to stabilize its binding
to various DNA substrates mimicking forked DNA
structures that are produced upon replication arrest
(Buss et al., 2008). Two other exonucleases, RecJ
and XseA (ExoVII subunit) were found localized
by SSB at chromosomal forks in B. subtilis. In E.
coli, the SSB partners RecQ and RecJ were found
to cooperate at stalled replication forks, degrading
nascent DNA prior to resumption of replication
(Courcelle and Hanawalt, 1999; Shereda et al.,
2007). The propensity of SSB proteins to recruit
various DNA helicases and exonucleases correlates
with the requirement for DNA unwinding and deg-
radation functions to remodel a disassembled fork.
Similarly, SSB’s interactions with RarA and RecO,
two proteins involved in the loading of RecA (the
central player in homologous recombination; see
Chapter 2) at arrested forks in E. coli (Lestini and
Michel, 2007), also illustrate the need for RecA-
mediated fork rescue in recovering replication after
accidental arrest. Finally, the physical interaction of
SSB with the essential polymerase DnaE, recently
shown to extend RNA primers laid down by DnaG
at forks (Sanders et al., 2010), suggests that DnaE
may also fulfil additional tasks in specialized repair
pathways. This dual role of DnaE as a DNA poly-
merase during replication initiated at oriC and in
replication restart at arrested forks is intriguing and
remains to be investigated.
18 | Murray et al.
Translesion DNA synthesis
Living cells are constantly exposed to endogenous
and exogenous DNA damaging agents and they
cope with DNA lesions by employing various DNA
repair pathways. In detrimental conditions E. coli
and B. subtilis cells activate a co-ordinated cellular
response, termed SOS, which controls the expres-
sion of several proteins involved in DNA repair,
recombination and mutagenesis (see Chapter
2) (Becherel and Fuchs, 1999; Courcelle et al.,
2001; Friedberg, 1995). Although efficient repair
processes act to remove most of the lesions from
DNA in an error-free manner, the persistence of
unrepaired damage may result in arrest of the DNA
replication fork as replicative DNA polymerases are
often unable to incorporate nucleotides opposite
non-instructive damaged bases.
The Y-family DNA polymerases
In SOS response-induced E. coli and B. subtilis cells,
mutagenesis at both damaged and undamaged
sites on the DNA template is increased (Becherel
and Fuchs, 1999; Duigou et al., 2004; Friedberg,
1995). This results from translesion DNA synthesis
(TLS), a process mediated by specialized DNA
polymerases that are capable of synthesis through
blocking DNA lesions (Friedberg et al., 2005;
Prakash et al., 2005). Lesion bypass is often error-
prone, thus producing mutations in the nascent
DNA strand opposite the damaged site. The vast
majority of TLS enzymes belong to the Y-family of
DNA polymerases, which is widely spread among
living organisms. Rev1p and Pol η in yeast, or Pol IV
and Pol V in E. coli, are prototypical members of this
family (Ohmori et al., 2001). The Y-polymerases
display low fidelity DNA synthesis, low processiv-
ity, and lack 3′–5′ proofreading exonuclease activity
(Kokoska et al., 2002; Kunkel and Bebenek, 2000).
These enzymes adopt the ‘right hand’ structure
typical of DNA polymerases, albeit with a more
flexible active site that can accommodate various
distorting lesions (Friedberg et al., 2001; Ling et al.,
2001). Depending on the context and nature of the
damaged site, bypass involves the recruitment of
one or more specialized DNA polymerases (Fried-
berg et al., 2005).
B. subtilis possesses two Y-family members,
PolY1 and PolY2, encoded by the yqjH and yqjW
genes, respectively (Duigou et al., 2004; Ohmori
et al., 2001). When overproduced PolY1 promotes
untargeted mutagenesis, a process that requires
its binding to the β-clamp (Duigou et al., 2004).
Furthermore, PolY1 acts in adaptive mutagenesis
(Sung et al., 2003). These characteristics are remi-
niscent of E. coli Pol IV, encoded by dinB, which
confers a mutator phenotype when overproduced
(Kim et al., 2001; Wagner et al., 1999; Wagner
and Nohmi, 2000) and also requires interaction
with the β-sliding clamp (Becherel et al., 2002;
Lenne-Samuel et al., 2002). The second B. subtilis
Y-polymerase, PolY2, is responsible for most UV-
induced mutagenesis in B. subtilis, and like PolY1
also promotes untargeted mutagenesis when over-
produced (Duigou et al., 2004). PolY2-mediated
mutagenesis on undamaged DNA requires the
presence of RecA in addition to interaction with
the β-clamp (Duigou et al., 2004, 2005). Based on
these properties, PolY2 appears to be the functional
homologue of E. coli Pol V. However, whereas Pol
V exists as a heterotrimeric complex termed
UmuCD2, B. subtilis PolY2 likely acts as a single
protein (no umuD homologue can be detected in
the B. subtilis genome) to carry out TLS across UV-
induced lesions. Evidence also suggests that both
PolY1 and PolY2 may participate in TLS during
sporulation, enhancing the survival of sporulating
cells exposed to genotoxic agents (Rivas-Castillo et
al., 2010).
Considering the mutagenic potential of Y-family
polymerases, clearly their intracellular levels must
be tightly controlled. In E. coli Pol IV is consti-
tutively expressed at a low level, Pol V is strongly
repressed under normal growth conditions, and
both polymerases are induced during the SOS
response (Courcelle et al., 2001). In M. tuberculosis
the two Y-family polymerases DinP and DinX are
expressed constitutively and are not up-regulated
after DNA damage (Boshoff et al., 2003; Brooks et
al., 2001). In B. subtilis PolY1 is expressed at a low
constitutive level and does not belong to the SOS
regulon, suggesting that it may act during normal
DNA replication, whereas PolY2 is expressed only
upon SOS induction (Duigou et al., 2004). It is
conceivable that PolY1 may be up-regulated under
specific environmental stress or at a specific stage
of development, consistent with its involvement
in adaptive mutagenesis (Sung et al., 2003). Thus,
it appears that different bacteria have evolved
different strategies to regulate the expression of
their Y-family polymerases, which might reflect
 Construction and Maintenance of a Replication Fork | 19

adaptation to their own environmental growth con-
ditions to minimize the detrimental effect of DNA
damage and increase their fitness and survival.
B. subtilis Pol I assists translesion
synthesis catalysed by Y-family
polymerases
Surprisingly, the vast majority (> 90%) of UV-
induced mutagenesis observed in B. subtilis upon
PolY2 overproduction depends on the presence of
catalytically proficient DNA polymerase I (Duigou
et al., 2005). It was found that Pol I directly
interacts with PolY2 in a yeast two-hybrid assay,
suggesting that Pol I may assist PolY2 in TLS across
UV lesions. The remaining (< 10%) untargeted
UV-induced mutagenesis mediated by PolY2 over-
production occurs in a Pol I-independent manner,
suggesting that for a minority of UV-damaged
sites, and for mutagenesis at undamaged sites,
PolY2 may perform TLS alone or be assisted by
another unidentified DNA polymerase. Similarly,
Pol I is required for the spontaneous mutagenesis
mediated by PolY1 overproduction and interacts
with the PolY1 N-terminal domain in a yeast two-
hybrid assay. The enzymatic activities of both Pol
I and PolY1 are required for TLS, suggesting that
they act in a co-ordinated manner to address muta-
tions during replication of DNA templates. Taken
together, these results indicate that the major
pathway for TLS in B. subtilis is a dual polymerase
mechanism involving PolY1 or PolY2 together with
DNA polymerase I (Duigou et al., 2005). Interest-
ingly, TLS in eukaryotes is often promoted by the
concerted action of two polymerases, one acting as
an ‘inserter’ at the damaged site and the other one
as an ‘extender’. However, such a TLS mechanism
is not present in E. coli (a comparison of the E. coli
and B. subtilis Y-family polymerases properties is
summarized in Table 1.3). Although the bypass
of some bulky lesions in particular DNA contexts
may require the concerted action of two DNA
polymerases in E. coli (Wagner et al., 2002), the
major pathway is a one-enzyme process carried out
by Pol V in the presence of RecA (Fujii and Fuchs,
2004; Pham et al., 2002; Tang et al., 2000). Pol IV-
mediated mutagenesis is similarly independent of
both Pol I and Pol II (Wagner and Nohmi, 2000).
Alignment of the B. subtilis Pol I sequence
with orthologues from various eubacteria reveals
that it belongs to a Gram-positive subfamily of
A-polymerases. Polymerases of this subfamily
have a vestigial 3′–5′ exonuclease active site which
lacks the residues involved in co-ordination of the
divalent cations required for exonuclease activity
(Duigou et al., 2005). Indeed it has been found that
B. subtilis Pol I is deficient for exonuclease activity
(Kiefer et al., 1997), which is consistent with its role
in TLS (i.e. – proofreading activity would abrogate

Table 1.3 E. coli and B. subtilis Y-family polymerases properties in growing cells
Expression Functional requirements
Involvement in
Polymerase Constitutive SOS mutagenesis in vivo RecA DnaN Pol I
Ec Pol IV Yes Yes Untargeted no yes no
Bs PolY1 Yes No Untargeted a
no yes yes
Ec Pol V No Yes Untargetedb yes yes no
UV-induced yesc yesd no
Bs PolY2 No Yes Untargeted yes yes no
UV-induced (nd)e nof yes

aRequirement for adaptive mutagenesis not determined (Sung et al., 2003).

bThe low fidelity of Pol V observed in vitro is proposed to be the mechanistic basis for chromosomal untargeted
mutagenesis (Maor-Shoshani and Livneh, 2002).
cThe involvement of RecA in Pol V catalysed TLS has been demonstrated in vitro (Reuven et al., 1999) and in vivo

(Sweasy et al., 1990).

dNot an absolute requirement in vitro (Pham et al., 2001).
eNot determined, due to the very high UV-sensitivity of the ΔrecA mutant (Fabret et al., 2002). Purified B. subtilis Pol

Y proteins are currently unavailable.

fBased on the absence of requirement for the integrity of the bacterial β-binding pentapeptide consensus for

UV-induced mutagenesis (Duigou et al., 2005).

20 | Murray et al.
translesion DNA synthesis by preventing the stable
incorporation of a mispaired nucleotide opposite
DNA lesions) (Khare and Eckert, 2002; Sagher
et al., 1994). E. coli Pol I, which is proficient for
3′–5′ proofreading activity, does not participate in
either spontaneous or UV-induced Pol IV-mediated
mutagenesis in vivo (Wagner and Nohmi, 2000).
However, a successful bypass event requires the
resumption of DNA synthesis by switching back
to the replicative polymerase. In E. coli Pol V syn-
thesizes a TLS-patch of a critical length that favours
further extension by the replicative polymerase,
rather than degradation (Fujii and Fuchs, 2004).
In B. subtilis a model has been proposed where
PolY1 or PolY2 carry out synthesis across the
lesion, then Pol I takes over to extend the synthesis
until the functional replisome resumes replication
in a manner similar to eukaryotic TLS (Fig. 1.3)
(Duigou et al., 2005). Altogether, these findings
support a new function of the A-family DNA poly-
merases that is likely conserved in Gram-positive
bacteria harbouring Pol I enzymes devoid of proof-
reading exonuclease activity.
New aspects of DNA polymerase I
functions
Deletion of DNA Pol I does not affect cell viability
in B. subtilis. The ypcP gene of B. subtilis encodes a
putative 5′–3′ exonuclease that is homologous to
the N-terminal domain of Pol I (38% identity).
The disruption of ypcP in a polA null background is
lethal, whereas it does not affect viability in a polA+
strain, suggesting that the redundant 5′–3′ exonu-
clease activity between Pol I and YpcP is essential
for cell survival (Duigou et al., 2005). Furthermore,
in a strain expressing a Pol I mutant deficient for
polymerase activity, the lack of YpcP did not reduce
cell survival, indicating that Pol I polymerase
activity is dispensable for cell survival. Pol I poly-
merase function was also found to be dispensable
in S. pneumoniae (Diaz et al., 1992). However, the
polymerase activity of B. subtilis Pol I is required for
the repair of MMS and UV-induced DNA damage
(Gass and Cozzarelli, 1973), as well as for the ini-
tiation of plasmid replication (Bruand et al., 1993).
Together with its role in the maturation of Okazaki
fragments (Tamanoi et al., 1977), Pol I appears to
be a multifunctional enzyme involved in several
pathways of DNA replication and repair.
In yeast two-hybrid assays B. subtilis Pol I
interacts with itself and with several other DNA
polymerases, including the two replicative DNA
polymerases PolC and DnaE. It also interacts with
HolB, the δ’ subunit of the clamp loader complex
(Noirot-Gros et al., 2002), and with the β clamp,
provided that a β-dimer interface is formed (Duigou
et al., 2005). All these interactions involve the same
internal specific interacting domain of Pol I that
comprises half of the vestigial 3′–5′ exonuclease and
part of the thumb domains. These interactions sug-
gest that Pol I could link several components of the
replication machinery in B. subtilis. It was suggested
that the mouse Rev1 protein, a member of the
Y-family of DNA polymerases, may also function as
a scaffold or a docking site to mediate polymerase
switching and/or to deliver the appropriate TLS
enzyme to the primer terminus opposite lesion sites
(Guo et al., 2003).
Termination of DNA replication
As in E. coli, the B. subtilis terminus region is organ-
ized as two oppositely oriented sets of ter sites
constituting a replication fork trap that allows
entry, but not escape, of incoming replication
forks (Duggin and Wake, 2002). The B. subtilis
termination protein RTP binds to the ter sites and
the RTP–ter complex blocks replication forks in a
polar manner. However, the termination systems
are markedly different as the E. coli Tus and B.
subtilis RTP termination proteins are unrelated
either in sequence or in their three-dimensional
structure, and the sequence of the ter sites share
no homology. In contrast to the monomeric Tus
protein that forms a 1:1 complex with its cognate
ter site, the RTP protein binds as two dimers to a
pair of overlapping half-sites within a ter element
and forms a structurally symmetrical RTP–ter
complex (Duggin and Wake, 2002). Each half-site
of ter binds an RTP dimer, one with high affinity
and the other with low affinity. A replication fork
is arrested only when it approaches the high affinity
(proximal) side of the complete RTP–ter complex,
suggesting that the differential binding affinity con-
tributes to the functional polarity of the terminator
complex. However, analysis of a series of mutant ter
sites revealed that the efficiency of replication fork
arrest in vivo is not directly correlated to the effec-
tive binding affinity of RTP at the proximal half-site,
indicating that this is not the only factor controlling
the polarity of fork arrest (Duggin et al., 2005).
 Construction and Maintenance of a Replication Fork | 21

C-family
replicative polymerase

5′
3′
β

Replication pause Replication arrest

without DNA lesion at endogenous lesion
(A) (B)

5′ 5′
3′ 3′ X

(A1) (B1)
PolY1 PolY1

5′ 5′
3′ 3′ X

(A2) (B2)
PolA PolA

5′ 5′
3′ 3′ X

Figure 1.3 A proposed mode of action of Pol I in PolY1-mediated untargeted mutagenesis in B. subtilis.
The replicative DNA polymerase (PolC) elongates a DNA strand at the replication fork. Under normal growth
conditions and in the absence of exogenous DNA damage treatment, fork progression can encounter a road
block, such as a protein–DNA complex (A) or an endogenous DNA lesion (B), that causes the disassembly of
the replication machinery. Pol Y1 lacks intrinsic proofreading activity and is prone to misincorporation during
replication at undamaged DNA templates. (A) PolY1 is recruited at stalled replication forks by interaction with
the β-sliding clamp through its C-terminal domain. PolY1-mediated DNA synthesis can proceed until erroneous
incorporation, resulting in replication arrest (step A1). Pol I (Pol A) subsequently extends from the mismatched
primer terminus, resulting in the fixation of the mutation (step A2). Recruitment of Pol I at the primer terminus
may be mediated by direct interaction with the PolY1 N-terminal domain. Resumption of normal replication
requires switching of the primer-template-bound polymerase back to PolC. (B) PolY1 is recruited at mismatched
primer termini by interaction with the β-sliding clamp. It incorporates a nucleotide opposite to the distorting or
non-instructive lesion, and is unable to proceed further (step B1). Pol I is recruited by interaction with the PolY1
N-terminal domain to assist further primer extension (step B2). Again, resumption of normal replication requires
replacement of Pol I by PolC on the primer-template. Note that it is not known whether PolY1 disengages from
the clamp during Pol I-mediated synthesis.
22 | Murray et al.
Furthermore, mutations in the C-terminal end of
RTP, which is proposed to contact the replication
machinery, do not affect the binding affinity for ter
but do reduce the capacity of the RTP–ter complex
to arrest replication forks in vivo, suggesting that an
interaction between RTP and the replisome could
enhance the efficiency of the replication block
(Duggin, 2006). Clearly, future studies will need to
combine structural and biochemical studies of RTP
bound to forked DNA in vitro with mutagenesis
and in vivo functional studies to elucidate whether
B. subtilis RTP-ter mediates polar termination by a
mechanism similar to the molecular mousetrap of
the E. coli Tus-ter termination complex (Mulcair et
al., 2006), or by a novel mechanism.
Integration of DNA replication with
other cellular processes in Bacillus
subtilis
Sporulation
In response to nutrient starvation B. subtilis
cells can enter a developmental pathway that
produces environmentally resistant endospores.
Activation of the pathway requires transduc-
tion of phosphate down a protein relay system
(KinA → Spo0F → Spo0B → Spo0A) culminating in
the phosphorylation and activation of Spo0A
(Piggot and Hilbert, 2004). Sporulation is charac-
terized by the formation of an asymmetrical septum
that produces a large mother cell and a smaller
forespore (Fig. 11.1). Each cell type requires one
copy of the genome in order to direct a distinct
program of chromosomal gene expression (Chap-
ter 11). Activation of the sporulation pathway
appears to require proper initiation of DNA repli-
cation, as mutations in the dnaA, dnaD and dnaB
genes inhibit sporulation in response to nutrient
starvation (Ireton and Grossman, 1994; Lemon
et al., 2000). Mutations that restore sporulation in
a dnaA mutant affect the sda gene (Burkholder et
al., 2001). Sda is an inhibitor of sporulation that is
overexpressed in replication initiation mutants and
sda transcription appears to be positively regulated
by DnaA (Breier and Grossman, 2009). Sda acts
by binding directly to the histidine kinase, KinA,
inhibiting its autophosphorylation and therefore
reducing levels of Spo0A~P (Cunningham and
Burkholder, 2009; Rowland et al., 2004). A GFP
reporter was used to detect sda promoter activity in
single cells during entry into sporulation, revealing
that the expression of sda occurred in a pulsatile
manner correlated with the initiation of DNA rep-
lication (Veening et al., 2009). The burst of sda at
the onset of DNA replication transiently inhibits
initiation of sporulation, as shown by the recipro-
cal relationship between the expression of sda
and spoIIA, a positive marker for early sporulation
(Veening et al., 2009). The control of sda expression
by DnaA constitutes a developmental checkpoint
by allowing cells to monitor initiation of DNA
replication and preventing attempts to sporulate
until chromosome replication has been completed
(Burkholder et al., 2001; Ruvolo et al., 2006; Veen-
ing et al., 2009).
More recently it has been found that DNA
replication is linked to sporulation in another way.
The arrangement of two sporulation phosphorelay
genes, spo0F located close to the origin and kinA
close to the terminus, leads to a transient gene
dosage imbalance following DNA replication ini-
tiation. The increase in spo0F copy-number leads to
an elevation of Spo0F that inhibits KinA and con-
sequently phosphorylation of Spo0A. Duplication
of kinA towards the end of chromosome replica-
tion causes KinA to accumulate and overcome
inhibition by Spo0F, thereby providing a pulse of
Spo0A~P once per cell cycle (Narula et al., 2015).
The mechanisms described above ensure that
entry into the sporulation developmental pathway
only occurs during the later stage of the cell divi-
sion cycle. Conversely, commitment to sporulate
induces multiple regulatory mechanisms that ensure
further DNA replication initiation is blocked. First,
Spo0A~P binds directly to oriC and inhibits DNA
duplex unwinding (Boonstra et al., 2013; Castilla-
Llorente et al., 2006). Second, Spo0A~P activates
the expression of the negative regulator, sirA. SirA
binds directly to domain I of DnaA, localizes with
the replisome in a DnaA-dependent manner, and
limits DnaA binding to oriC ( Jameson et al., 2014;
Rahn-Lee et al., 2009, 2011; Wagner et al., 2009);
however, the precise regulatory mechanism used
by SirA to inhibit DNA replication initiation is not
fully understood.
Transcription
In vegetative B. subtilis cells perturbations of DNA
replication, caused by treatments with a DNA poly-
merase inhibitor or DNA-damaging agents, induce

global transcriptional responses that enhance cell
survival. The SOS response is triggered by the bind-
ing of the recombination protein, RecA, to ssDNA,
followed by the stimulation of autocleavage of the
transcriptional repressor, LexA (also called DinR;
see Chapter 2) (Friedberg, 1995). RecA appears
to be a global regulator as it affects the expression
of approximately 450 genes amongst a total of 668
genes that are induced in response to treatment
with the PolC inhibitor p-hydroxyphenylazo-uracil
(HPUra) (Goranov et al., 2006). DNA microarray
analysis of mRNA levels in B. subtilis indicates that
26 operons, containing at least 63 genes, are directly
controlled by LexA and are induced after replication
arrest or DNA damage (Au et al., 2005; Goranov et
al., 2006). Additionally, induction of sda following
replication arrest inhibits the activation of Spo0A,
thereby affecting the expression of about 100 genes
(Burkholder et al., 2001; Molle et al., 2003; Row-
land et al., 2004). The transcriptional responses
to perturbations in DNA replication couple DNA
replication to a spectrum of cellular processes
including sporulation, amino acid and nucleotide
biosynthesis, translation, ribosome assembly and
response to oxidation (Goranov et al., 2005).
Interestingly, the inhibition of the elongation
step of DNA replication elicits a distinct transcrip-
tional response that affects the expression of over
100 genes but is independent of RecA and Sda
(Goranov et al., 2005). The majority of these genes
respond similarly to the inhibition of either elonga-
tion or initiation of DNA replication, suggesting
that the mechanism controlling the expression
of these genes does not sense arrested replication
forks. Many of the recA- and sda-independent
transcriptional responses (~56 genes) appear to be
mediated by the replication initiator, DnaA, which
can itself act directly as a transcription regulator. For
example, it has been shown that DnaA binds to the
promoter region of the yllB-ylxA-ftsL-pbpB operon
in vivo and that the mRNA levels of these four
genes decrease upon replication arrest (Goranov et
al., 2005). Both ftsL and pbpB encode essential cell
division proteins (Fig. 4.4) (Daniel et al., 1996).
Artificial constitutive expression of ftsL-pbpB under
conditions of replication arrest allows cell division
but decreases cell viability, indicating that the inhi-
bition of ftsL-pbpB transcription by DnaA serves to
couple cell division with DNA replication in order
Construction and Maintenance of a Replication Fork | 23
to maintain a productive cell cycle (Goranov et al.,
2005).
Cell growth
In most bacteria nutrient limitation leads to the
accumulation of the alarmone (p)ppGpp and
provokes the stringent control of various cellular
processes (Potrykus and Cashel, 2008) (Figs. 15.1
and 15.2). As in E. coli, DNA replication is under
stringent control in Bacillus (Seror et al., 1991).
Studies have revealed that in B. subtilis the stringent
response regulates DNA replication elongation
through the binding of (p)ppGpp within the active
site of primase (DnaG), leading to replication
fork arrest (Gourse and Keck, 2007; Rymer et al.,
2012; Wang et al., 2007). This is in contrast to E.
coli where the stringent response was shown to
trigger replication arrest at initiation (Ferullo and
Lovett, 2008), although recent studies suggest
(p)ppGpp may weakly inhibit DNA replication
elongation through affects on primase (Denapoli et
al., 2013; Maciag et al., 2010). Interestingly, RecA
is not recruited to replication forks stalled by (p)
ppGpp, indicating that the arrested forks are not
disrupted (Wang et al., 2007). Thus, in B. subtilis the
nucleotide (p)ppGpp acts as a sensor mechanism
in nutrient-limited cells that prevents replication-
fork progression yet preserves fork integrity.
Further supporting the notion that DNA rep-
lication is co-ordinated with cell growth, several
distinct connections between DNA replication
and essential cellular activities have been docu-
mented, including respiration (ndh), central carbon
metabolism (gapA, pykA, pdhB), fatty acid synthe-
sis (fabHA), phospholipid synthesis (plsC, pgsA),
and protein synthesis (rpmJ, rplA, rplW, rpsU)
( Janniere et al., 2007; Murray and Koh, 2014). Dis-
ruption of specific biosynthetic pathways by gene
deletion or enzyme depletion caused a decrease
in the frequency of DNA replication initiation.
Although the regulatory mechanisms remain
unknown, genetic evidence indicates that several of
the pathways require DnaA-dependent initiation at
oriC (Murray and Koh, 2014). Similar results have
been found in E. coli, suggesting that coordination
of DNA replication with central metabolic activities
is widely conserved (Maciag et al., 2011; Sekimizu
and Kornberg, 1988; Xia and Dowhan, 1995).
24 | Murray et al.
Conclusions and future
perspectives
The level of detail at which DNA replication is
understood in B. subtilis is second only to E. coli
among prokaryotes. The comparison between
both organisms reveals similarities in the overall
architecture of the replisome, in the ways DNA
replication is initiated at the chromosomal origin
and re-initiated at arrested replication forks, and
how this results in the spatially and temporally
controlled loading of the replicative helicase onto
the single-stranded lagging-strand template. The
comparison also reveals many striking differences
that appear to be highly conserved among Gram-
positive bacteria.
As in E. coli, the B. subtilis replicase comprises a
Pol III enzyme, a β2 and a DnaX complex, as well as
a hexameric replicative helicase. However, B. subtilis
makes use of two helicase co-loaders (vs. one in E.
coli), as observed in eukaryotic cells (Velten et al.,
2003). Another difference lies in the involvement
of three different players (DnaD, DnaB and DnaI)
in PriA-dependent replication restart (Bruand et
al., 2001). The most remarkable divergence is the
requirement for two essential replicases in B. sub-
tilis, PolC and DnaE, instead of the single Pol III
enzyme in E. coli; this points to a major mechanistic
difference in chromosomal replication between the
two bacteria (Dervyn et al., 2001; Sanders et al.,
2010). DnaE, through its ability to elongate RNA
primers and to work synergistically with PolC on
the lagging strand, displays functional similarities
with the eukaryotic Pol α. DnaE is endowed with
low fidelity, is capable of lesion bypass, and is
recruited to the replication fork via an interaction
with SSB (rather than DnaN as with PolC). The
significance of selective SSB-mediated targeting of
DnaE at replication forks remains to be functionally
explained.
B. subtilis DNA polymerase I processes Okazaki
fragments, participates in the repair of DNA damage
and plasmid replication, and assists in translesion
synthesis mediated by the Y-polymerases. Pol I
lacks an exonuclease proofreading activity, in keep-
ing with its role in TLS, but counter-productive
to error-free DNA synthesis required for DNA
replication. Does B. subtilis Pol I mediate DNA
synthesis in vivo, or does it fulfil a structural role,
possibly through its interactions with PolC and
DnaE polymerases?
DnaA-dependent initiation is negatively regu-
lated by YabA and by Soj, and both appear to target
DnaA filament formation. It is unclear exactly how
this regulation is achieved, or whether these factors
target distinct DnaA filaments (i.e. – formed on
double-stranded DNA or a single DNA strand).
Similarly, it is unclear how DnaA–box clusters
regulate DnaA activity, and whether a specific
arrangement of binding sites is required. Further
genetic, biochemical, and structural studies will be
required to address these questions.
B. subtilis is the model bacterial organism in
which the replication factory has been discovered
and where the subcellular localization of most
replication proteins is known. A major challenge
is to understand how such a replication factory
is constructed, maintained, and disassembled
throughout the cell cycle. In combination, the sub-
cellular localization of proteins, the transcriptional
responses to perturbations of replication, and the
interaction network of replication proteins will help
to identify the proteins that act to co-ordinate DNA
replication with other cellular processes.
Acknowledgements
We thank all of the past and present members of our
laboratories for their contributions to the under-
standing of various aspects of DNA replication, and
for many stimulating discussions. We apologize to
those whose work has not been cited adequately
because of space limitations.
References
Au, N., Kuester-Schoeck, E., Mandava, V., Bothwell, L.E.,
Canny, S.P., Chachu, K., Colavito, S.A., Fuller, S.N.,
Groban, E.S., Hensley, L.A., et al. (2005). Genetic
composition of the Bacillus subtilis SOS system. J.
Bacteriol. 187, 7655–7666. http://dx.doi.org/10.1128/
JB.187.22.7655-7666.2005
Bailey, S., Eliason, W.K., and Steitz, T.A. (2007). Structure of
hexameric DnaB helicase and its complex with a domain
of DnaG primase. Science 318, 459–463. http://dx.doi.
org/10.1126/science.1147353
Barnes, M.H., Hammond, R.A., Kennedy, C.C., Mack,
S.L., and Brown, N.C. (1992). Localization of the
exonuclease and polymerase domains of Bacillus subtilis
DNA polymerase III. Gene 111, 43–49.
Barnes, M.H., Leo, C.J., and Brown, N.C. (1998). DNA
polymerase III of Gram-positive eubacteria is a zinc
metalloprotein conserving an essential finger-like
domain. Biochemistry 37, 15254–15260. http://dx.doi.
org/10.1021/bi981113m
Becherel, O.J., and Fuchs, R.P. (1999). SOS mutagenesis
results from up-regulation of translesion synthesis. J.

Mol. Biol. 294, 299–306. http://dx.doi.org/10.1006/
jmbi.1999.3272
Becherel, O.J., Fuchs, R.P., and Wagner, J. (2002). Pivotal
role of the beta-clamp in translesion DNA synthesis and
mutagenesis in E. coli cells. DNA. Repair 1, 703–708.
Benkovic, S.J., Valentine, A.M., and Salinas, F. (2001).
Replisome-mediated DNA replication. Annu. Rev.
Biochem. 70, 181–208.
Berghuis, B.A., Dulin, D., Xu, Z.Q., van Laar, T., Cross, B.,
Janissen, R., Jergic, S., Dixon, N.E., Depken, M., and
Dekker, N.H. (2015). Strand separation establishes a
sustained lock at the Tus-Ter replication fork barrier. Nat.
Chem. Biol. 11, 579–585. http://dx.doi.org/10.1038/
nchembio.1857
Berka, R.M., Hahn, J., Albano, M., Draskovic, I., Persuh, M.,
Cui, X., Sloma, A., Widner, W., and Dubnau, D. (2002).
Microarray analysis of the Bacillus subtilis K-state:
genome-wide expression changes dependent on ComK.
Mol. Microbiol. 43, 1331–1345.
Bird, L.E., Pan, H., Soultanas, P., and Wigley, D.B. (2000).
Mapping protein-protein interactions within a stable
complex of DNA primase and DnaB helicase from
Bacillus stearothermophilus. Biochemistry 39, 171–182.
Bird, L.E., and Wigley, D.B. (1999). The Bacillus
stearothermophilus replicative helicase: cloning,
overexpression and activity. Biochim. Biophys. Acta.
1444, 424–428.
Boeneman, K., and Crooke, E. (2005). Chromosomal
replication and the cell membrane. Curr. Opin.
Microbiol. 8, 143–148. http://dx.doi.org/10.1016/j.
mib.2005.02.006
Boonstra, M., de Jong, I.G., Scholefield, G., Murray,
H., Kuipers, O.P., and Veening, J.W. (2013). Spo0A
regulates chromosome copy number during sporulation
by directly binding to the origin of replication in Bacillus
subtilis. Mol. Microbiol. 87, 925–938.
Boshoff, H.I., Reed, M.B., Barry, C.E., and Mizrahi, V.
(2003). DnaE2 polymerase contributes to in vivo survival
and the emergence of drug resistance in Mycobacterium
tuberculosis. Cell 113, 183–193.
Boye, E., Løbner-Olesen, A., and Skarstad, K. (2000).
Limiting DNA replication to once and only once.
EMBO Rep. 1, 479–483. http://dx.doi.org/10.1093/
embo-reports/kvd116
Bravo, A., Serrano-Heras, G., and Salas, M. (2005).
Compartmentalization of prokaryotic DNA replication.
FEMS Microbiol. Rev. 29, 25–47. http://dx.doi.
org/10.1016/j.femsre.2004.06.003
Breier, A.M., and Grossman, A.D. (2009). Dynamic
association of the replication initiator and transcription
factor DnaA with the Bacillus subtilis chromosome
during replication stress. J. Bacteriol. 191, 486–493.
Brooks, P.C., Movahedzadeh, F., and Davis, E.O. (2001).
Identification of some DNA damage-inducible genes of
Mycobacterium tuberculosis: apparent lack of correlation
with LexA binding. J. Bacteriol. 183, 4459–4467. http://
dx.doi.org/10.1128/JB.183.15.4459-4467.2001
Bruand, C., and Ehrlich, S.D. (1995). The Bacillus subtilis
dnaI gene is part of the dnaB operon. Microbiology 141,
1199–1200. http://dx.doi.org/10.1099/13500872-
141-5-1199
Construction and Maintenance of a Replication Fork | 25
Bruand, C., Ehrlich, S.D., and Jannière, L. (1995).
Primosome assembly site in Bacillus subtilis. EMBO J.
14, 2642–2650.
Bruand, C., Farache, M., McGovern, S., Ehrlich, S.D., and
Polard, P. (2001). DnaB, DnaD and DnaI proteins are
components of the Bacillus subtilis replication restart
primosome. Mol. Microbiol. 42, 245–255.
Bruand, C., Le Chatelier, E., Ehrlich, S.D., and Jannière, L.
(1993). A fourth class of theta-replicating plasmids: the
pAM beta 1 family from gram-positive bacteria. Proc.
Natl. Acad. Sci. U.S.A. 90, 11668–11672.
Bruand, C., Velten, M., McGovern, S., Marsin, S., Sérèna,
C., Ehrlich, S.D., and Polard, P. (2005). Functional
interplay between the Bacillus subtilis DnaD and DnaB
proteins essential for initiation and re-initiation of DNA
replication. Mol. Microbiol. 55, 1138–1150. http://
dx.doi.org/10.1111/j.1365-2958.2004.04451.x
Bruck, I., Georgescu, R.E., and O’Donnell, M. (2005).
Conserved interactions in the Staphylococcus aureus
DNA PolC chromosome replication machine. J. Biol.
Chem. 280, 18152–18162. http://dx.doi.org/10.1074/
jbc.M413595200
Bruck, I., Goodman, M.F., and O’Donnell, M. (2003). The
essential C family DnaE polymerase is error-prone and
efficient at lesion bypass. J. Biol. Chem. 278, 44361–
44368. http://dx.doi.org/10.1074/jbc.M308307200
Bruck, I., and O’Donnell, M. (2000). The DNA replication
machine of a gram-positive organism. J. Biol. Chem.
275, 28971–28983. http://dx.doi.org/10.1074/jbc.
M003565200
Burkholder, W.F., Kurtser, I., and Grossman, A.D. (2001).
Replication initiation proteins regulate a developmental
checkpoint in Bacillus subtilis. Cell 104, 269–279.
Buss, J.A., Kimura, Y., and Bianco, P.R. (2008). RecG
interacts directly with SSB: implications for stalled
replication fork regression. Nucleic Acids Res. 36,
7029–7042. http://dx.doi.org/10.1093/nar/gkn795
Camara, J.E., Breier, A.M., Brendler, T., Austin, S., Cozzarelli,
N.R., and Crooke, E. (2005). Hda inactivation of DnaA is
the predominant mechanism preventing hyperinitiation
of Escherichia coli DNA replication. EMBO Rep. 6, 736–
741. http://dx.doi.org/10.1038/sj.embor.7400467
Campbell, J.L., and Kleckner, N. (1990). E. coli oriC
and the dnaA gene promoter are sequestered from
dam methyltransferase following the passage of the
chromosomal replication fork. Cell 62, 967–979.
Carneiro, M.J., Zhang, W., Ioannou, C., Scott, D.J.,
Allen, S., Roberts, C.J., and Soultanas, P. (2006). The
DNA-remodelling activity of DnaD is the sum of
oligomerization and DNA-binding activities on separate
domains. Mol. Microbiol. 60, 917–924. http://dx.doi.
org/10.1111/j.1365-2958.2006.05152.x
Castilla-Llorente, V., Muñoz-Espín, D., Villar, L., Salas, M.,
and Meijer, W.J. (2006). Spo0A, the key transcriptional
regulator for entrance into sporulation, is an inhibitor
of DNA replication. EMBO J. 25, 3890–3899. http://
dx.doi.org/10.1038/sj.emboj.7601266
Chintakayala, K., Larson, M.A., Grainger, W.H., Scott, D.J.,
Griep, M.A., Hinrichs, S.H., and Soultanas, P. (2007).
Domain swapping reveals that the C- and N-terminal
domains of DnaG and DnaB, respectively, are functional
homologues. Mol. Microbiol. 63, 1629–1639. http://
dx.doi.org/10.1111/j.1365-2958.2007.05617.x
26 | Murray et al.
Chintakayala, K., Larson, M.A., Griep, M.A., Hinrichs,
S.H., and Soultanas, P. (2008). Conserved residues of
the C-terminal p16 domain of primase are involved in
modulating the activity of the bacterial primosome. Mol.
Microbiol. 68, 360–371. http://dx.doi.org/10.1111/
j.1365-2958.2008.06155.x
Chintakayala, K., Machón, C., Haroniti, A., Larson, M.A.,
Hinrichs, S.H., Griep, M.A., and Soultanas, P. (2009).
Allosteric regulation of the primase (DnaG) activity
by the clamp-loader (tau) in vitro. Mol. Microbiol. 72,
537–549.
Cho, E., Ogasawara, N., and Ishikawa, S. (2008). The
functional analysis of YabA, which interacts with DnaA
and regulates initiation of chromosome replication in
Bacillus subtils. Genes. Genet. Syst. 83, 111–125.
Costes, A., Lecointe, F., McGovern, S., Quevillon-Cheruel,
S., and Polard, P. (2010). The C-terminal domain of the
bacterial SSB protein acts as a DNA maintenance hub
at active chromosome replication forks. PLoS Genet.
6, e1001238. http://dx.doi.org/10.1371/journal.
pgen.1001238
Courcelle, J., Ganesan, A.K., and Hanawalt, P.C. (2001).
Therefore, what are recombination proteins there for?
Bioessays 23, 463–470.
Courcelle, J., and Hanawalt, P.C. (1999). RecQ and RecJ
process blocked replication forks prior to the resumption
of replication in UV-irradiated Escherichia coli. Mol. Gen.
Genet. 262, 543–551.
Cunningham, K.A., and Burkholder, W.F. (2009). The
histidine kinase inhibitor Sda binds near the site
of autophosphorylation and may sterically hinder
autophosphorylation and phosphotransfer to
Spo0F. Mol. Microbiol. 71, 659–677. http://dx.doi.
org/10.1111/j.1365-2958.2008.06554.x
Dalrymple, B.P., Kongsuwan, K., Wijffels, G., Dixon, N.E.,
and Jennings, P.A. (2001). A universal protein-protein
interaction motif in the eubacterial DNA replication and
repair systems. Proc. Natl. Acad. Sci. U.S.A. 98, 11627–
11632. http://dx.doi.org/10.1073/pnas.191384398
Daniel, R.A., Williams, A.M., and Errington, J. (1996).
A complex four-gene operon containing essential cell
division gene pbpB in Bacillus subtilis. J. Bacteriol. 178,
2343–2350.
Davey, M.J., and O’Donnell, M. (2003). Replicative helicase
loaders: ring breakers and ring makers. Curr. Biol. 13,
R594–6.
Denapoli, J., Tehranchi, A.K., and Wang, J.D. (2013).
Dose-dependent reduction of replication elongation rate
by (p)ppGpp in Escherichia coli and Bacillus subtilis. Mol.
Microbiol. 88, 93–104. http://dx.doi.org/10.1111/
mmi.12172
Dervyn, E., Suski, C., Daniel, R., Bruand, C., Chapuis, J.,
Errington, J., Jannière, L., and Ehrlich, S.D. (2001).
Two essential DNA polymerases at the bacterial
replication fork. Science 294, 1716–1719. http://dx.doi.
org/10.1126/science.1066351
Díaz, A., Lacks, S.A., and López, P. (1992). The 5′ to 3’
exonuclease activity of DNA polymerase I is essential for
Streptococcus pneumoniae. Mol. Microbiol. 6, 3009–3019.
Dohrmann, P.R., and McHenry, C.S. (2005). A bipartite
polymerase-processivity factor interaction: only the
internal beta binding site of the alpha subunit is required
for processive replication by the DNA polymerase III
holoenzyme. J. Mol. Biol. 350, 228–239.
Duderstadt, K.E., Chuang, K., and Berger, J.M. (2011). DNA
stretching by bacterial initiators promotes replication
origin opening. Nature 478, 209–213. http://dx.doi.
org/10.1038/nature10455
Duderstadt, K.E., Mott, M.L., Crisona, N.J., Chuang, K.,
Yang, H., and Berger, J.M. (2010). Origin remodeling
and opening in bacteria rely on distinct assembly states
of the DnaA initiator. J. Biol. Chem. 285, 28229–28239.
http://dx.doi.org/10.1074/jbc.M110.147975
Duggin, I.G. (2006). DNA replication fork arrest by
the Bacillus subtilis RTP-DNA complex involves a
mechanism that is independent of the affinity of
RTP-DNA binding. J. Mol. Biol. 361, 1–6. http://dx.doi.
org/10.1016/j.jmb.2006.06.013
Duggin, I.G., Matthews, J.M., Dixon, N.E., Wake, R.G., and
Mackay, J.P. (2005). A complex mechanism determines
polarity of DNA replication fork arrest by the replication
terminator complex of Bacillus subtilis. J. Biol. Chem.
280, 13105–13113.
Duggin, I.G., and Wake, R.G. (2002). Termination of
chromosome replication. In Bacillus subtilis and its
closest relatives: from genes to cells, A.L. Sonenshein,
J.A. Hoch, and R. Losick, eds. ASM Press, American
Society for Microbiology, Washington, D.C., 87–95.
Duigou, S., Ehrlich, S.D., Noirot, P., and Noirot-Gros, M.F.
(2004). Distinctive genetic features exhibited by the
Y-family DNA polymerases in Bacillus subtilis. Mol.
Microbiol. 54, 439–451. http://dx.doi.org/10.1111/
j.1365-2958.2004.04259.x
Duigou, S., Ehrlich, S.D., Noirot, P., and Noirot-Gros, M.F.
(2005). DNA polymerase I acts in translesion synthesis
mediated by the Y-polymerases in Bacillus subtilis. Mol.
Microbiol. 57, 678–690. http://dx.doi.org/10.1111/
j.1365-2958.2005.04725.x
Elshenawy, M.M., Jergic, S., Xu, Z.Q., Sobhy, M.A.,
Takahashi, M., Oakley, A.J., Dixon, N.E., and Hamdan,
S.M. (2015). Replisome speed determines the efficiency
of the Tus-Ter replication termination barrier. Nature
525, 394–398. http://dx.doi.org/10.1038/nature14866
Erzberger, J.P., Mott, M.L., and Berger, J.M. (2006).
Structural basis for ATP-dependent DnaA assembly and
replication-origin remodeling. Nat. Struct. Mol. Biol. 13,
676–683. http://dx.doi.org/10.1038/nsmb1115
Erzberger, J.P., Pirruccello, M.M., and Berger, J.M. (2002).
The structure of bacterial DnaA: implications for general
mechanisms underlying DNA replication initiation.
EMBO J. 21, 4763–4773.
Fabret, C., Ehrlich, S.D., and Noirot, P. (2002). A new
mutation delivery system for genome-scale approaches
in Bacillus subtilis. Mol. Microbiol. 46, 25–36.
Felicori, L., Jameson, K.H., Roblin, P., Fogg, M.J.,
Garcia-Garcia, T., Ventroux, M., Cherrier, M.V., Bazin, A.,
Noirot, P., Wilkinson, A.J., et al. (2016). Tetramerization
and interdomain flexibility of the replication initiation
controller YabA enables simultaneous binding to
multiple partners. Nucleic Acids Res. 44, 449–463.
Ferullo, D.J., and Lovett, S.T. (2008). The stringent response
and cell cycle arrest in Escherichia coli. PLoS Genet.
4, e1000300. http://dx.doi.org/10.1371/journal.
pgen.1000300

Friedberg, E.C., Fischhaber, P.L., and Kisker, C. (2001).
Error-prone DNA polymerases: novel structures and the
benefits of infidelity. Cell 107, 9–12.
Friedberg, E.C., Lehmann, A.R., and Fuchs, R.P. (2005).
Trading places: how do DNA polymerases switch during
translesion DNA synthesis? Mol. Cell 18, 499–505.
http://dx.doi.org/10.1016/j.molcel.2005.03.032
Friedberg, E.C., Walker, G.C., and Wolfram, S. (1995). SOS
responses and DNA damage tolerance in prokaryotes. In
DNA Repair and Mutagenesis, E.C. Friedberg, Walker
G. C., and Wolfram S., ed.,407–453.
Fujii, S., and Fuchs, R.P. (2004). Defining the position
of the switches between replicative and bypass DNA
polymerases. EMBO J. 23, 4342–4352. http://dx.doi.
org/10.1038/sj.emboj.7600438
Fujimitsu, K., Senriuchi, T., and Katayama, T. (2009).
Specific genomic sequences of E. coli promote
replicational initiation by directly reactivating
ADP-DnaA. Genes Dev. 23, 1221–1233. http://dx.doi.
org/10.1101/gad.1775809
Gass, K.B., and Cozzarelli, N.R. (1973). Further genetic
and enzymological characterization of the three Bacillus
subtilis deoxyribonucleic acid polymerases. J. Biol.
Chem. 248, 7688–7700.
Geertsema, H.J., and van Oijen, A.M. (2013). A
single-molecule view of DNA replication: the dynamic
nature of multi-protein complexes revealed. Curr. Opin.
Struct. Biol. 23, 788–793. http://dx.doi.org/10.1016/j.
sbi.2013.06.018
Goranov, A.I., Breier, A.M., Merrikh, H., and Grossman,
A.D. (2009). YabA of Bacillus subtilis controls
DnaA-mediated replication initiation but not the
transcriptional response to replication stress. Mol.
Microbiol. 74, 454–466.
Goranov, A.I., Katz, L., Breier, A.M., Burge, C.B., and
Grossman, A.D. (2005). A transcriptional response to
replication status mediated by the conserved bacterial
replication protein DnaA. Proc. Natl. Acad. Sci. U.S.A.
102, 12932–12937. http://dx.doi.org/10.1073/
pnas.0506174102
Goranov, A.I., Kuester-Schoeck, E., Wang, J.D., and
Grossman, A.D. (2006). Characterization of the global
transcriptional responses to different types of DNA
damage and disruption of replication in Bacillus subtilis. J.
Bacteriol. 188, 5595–5605. http://dx.doi.org/10.1128/
JB.00342-06
Gorbalenya, A.E., and Koonin, E.V. (1993). Helicases: amino
acid sequence comparisons and structure-function
relationship. Curr. Opin. Struct. Biol. 3, 419–429.
Gourse, R.L., and Keck, J.L. (2007). Magic spots cast a spell
on DNA primase. Cell 128, 823–824. http://dx.doi.
org/10.1016/j.cell.2007.02.020
Grainger, W.H., Machón, C., Scott, D.J., and Soultanas,
P. (2010). DnaB proteolysis in vivo regulates
oligomerization and its localization at oriC in Bacillus
subtilis. Nucleic Acids Res. 38, 2851–2864. http://
dx.doi.org/10.1093/nar/gkp1236
Gross, J.D., Karamata, D., and Hempstead, P.G. (1968).
Temperature-sensitive mutants of B. subtilis defective in
DNA synthesis. Cold Spring Harbor Symp. Quant. Biol.
33, 307–312.
Gu, S., Li, W., Zhang, H., Fleming, J., Yang, W., Wang, S.,
Wei, W., Zhou, J., Zhu, G., Deng, J., et al. (2016). The
Construction and Maintenance of a Replication Fork | 27
β2 clamp in the Mycobacterium tuberculosis DNA
polymerase III αβ2ε replicase promotes polymerization
and reduces exonuclease activity. Sci. Rep. 6, 18418.
Guo, C., Fischhaber, P.L., Luk-Paszyc, M.J., Masuda, Y.,
Zhou, J., Kamiya, K., Kisker, C., and Friedberg, E.C.
(2003). Mouse Rev1 protein interacts with multiple
DNA polymerases involved in translesion DNA
synthesis. EMBO J. 22, 6621–6630. http://dx.doi.
org/10.1093/emboj/cdg626
Hahn, J., Maier, B., Haijema, B.J., Sheetz, M., and Dubnau,
D. (2005). Transformation proteins and DNA uptake
localize to the cell poles in Bacillus subtilis. Cell 122,
59–71. http://dx.doi.org/10.1016/j.cell.2005.04.035
Hamdan, S.M., and van Oijen, A.M. (2010). Timing,
coordination, and rhythm: acrobatics at the DNA
replication fork. J. Biol. Chem. 285, 18979–18983.
http://dx.doi.org/10.1074/jbc.R109.022939
Hardy, C.D., Crisona, N.J., Stone, M.D., and Cozzarelli, N.R.
(2004). Disentangling DNA during replication: a tale
of two strands. Philos. Trans. R. Soc. Lond. B. Biol. Sci.
359, 39–47. http://dx.doi.org/10.1098/rstb.2003.1363
Haroniti, A., Anderson, C., Doddridge, Z., Gardiner, L.,
Roberts, C.J., Allen, S., and Soultanas, P. (2004). The
clamp-loader-helicase interaction in Bacillus. Atomic
force microscopy reveals the structural organisation
of the DnaB-tau complex in Bacillus. J. Mol. Biol. 336,
381–393.
Haroniti, A., Till, R., Smith, M.C., and Soultanas, P. (2003).
Clamp-loader-helicase interaction in Bacillus. Leucine
381 is critical for pentamerization and helicase binding
of the Bacillus tau protein. Biochemistry 42, 10955–
10964. http://dx.doi.org/10.1021/bi034955g
Hayashi, M., Ogura, Y., Harry, E.J., Ogasawara, N., and
Moriya, S. (2005). Bacillus subtilis YabA is involved in
determining the timing and synchrony of replication
initiation. FEMS Microbiol. Lett. 247, 73–79. http://
dx.doi.org/10.1016/j.femsle.2005.04.028
Heller, R.C., and Marians, K.J. (2005a). The disposition
of nascent strands at stalled replication forks dictates
the pathway of replisome loading during restart.
Mol. Cell 17, 733–743. http://dx.doi.org/10.1016/j.
molcel.2005.01.019
Heller, R.C., and Marians, K.J. (2005b). Unwinding of the
nascent lagging strand by Rep and PriA enables the
direct restart of stalled replication forks. J. Biol. Chem.
280, 34143–34151. http://dx.doi.org/10.1074/jbc.
M507224200
Heller, R.C., and Marians, K.J. (2006). Replication
fork reactivation downstream of a blocked nascent
leading strand. Nature 439, 557–562. http://dx.doi.
org/10.1038/nature04329
Imai, Y., Ogasawara, N., Ishigo-Oka, D., Kadoya, R., Daito,
T., and Moriya, S. (2000). Subcellular localization of
Dna-initiation proteins of Bacillus subtilis: evidence that
chromosome replication begins at either edge of the
nucleoids. Mol. Microbiol. 36, 1037–1048.
Indiani, C., McInerney, P., Georgescu, R., Goodman,
M.F., and O’Donnell, M. (2005). A sliding-clamp
toolbelt binds high- and low-fidelity DNA polymerases
simultaneously. Mol. Cell 19, 805–815. http://dx.doi.
org/10.1016/j.molcel.2005.08.011
Inoue, R., Kaito, C., Tanabe, M., Kamura, K., Akimitsu,
N., and Sekimizu, K. (2001). Genetic identification
28 | Murray et al.
of two distinct DNA polymerases, DnaE and PolC,
that are essential for chromosomal DNA replication
in Staphylococcus aureus. Mol. Genet. Genomics. 266,
564–571. http://dx.doi.org/10.1007/s004380100564
Ireton, K., and Grossman, A.D. (1994). A developmental
checkpoint couples the initiation of sporulation to DNA
replication in Bacillus subtilis. EMBO J. 13, 1566–1573.
Ishida, T., Akimitsu, N., Kashioka, T., Hatano, M., Kubota,
T., Ogata, Y., Sekimizu, K., and Katayama, T. (2004).
DiaA, a novel DnaA-binding protein, ensures the timely
initiation of Escherichia coli chromosome replication.
J. Biol. Chem. 279, 45546–45555. http://dx.doi.
org/10.1074/jbc.M402762200
Ishigo-Oka, D., Ogasawara, N., and Moriya, S. (2001).
DnaD protein of Bacillus subtilis interacts with DnaA,
the initiator protein of replication. J. Bacteriol. 183,
2148–2150. http://dx.doi.org/10.1128/JB.183.6.2148-
2150.2001
Ishikawa, S., Ogura, Y., Yoshimura, M., Okumura, H., Cho,
E., Kawai, Y., Kurokawa, K., Oshima, T., and Ogasawara,
N. (2007). Distribution of stable DnaA-binding sites on
the Bacillus subtilis genome detected using a modified
ChIP-chip method. DNA Res. 14, 155–168. http://
dx.doi.org/10.1093/dnares/dsm017
Jameson, K.H., Rostami, N., Fogg, M.J., Turkenburg, J.P.,
Grahl, A., Murray, H., and Wilkinson, A.J. (2014).
Structure and interactions of the Bacillus subtilis
sporulation inhibitor of DNA replication, SirA, with
domain I of DnaA. Mol. Microbiol. 93, 975–991. http://
dx.doi.org/10.1111/mmi.12713
Jannière, L., Canceill, D., Suski, C., Kanga, S., Dalmais, B.,
Lestini, R., Monnier, A.F., Chapuis, J., Bolotin, A., Titok,
M., et al. (2007). Genetic evidence for a link between
glycolysis and DNA replication. PLoS One 2, e447.
Johnson, A., and O’Donnell, M. (2005). Cellular DNA
replicases: components and dynamics at the replication
fork. Annu. Rev. Biochem. 74, 283–315.
Karamata, D., and Gross, J.D. (1970). Isolation and genetic
analysis of temperature-sensitive mutants of B. subtilis
defective in DNA synthesis. Mol. Gen. Genet. 108,
277–287.
Kasho, K., and Katayama, T. (2013). DnaA binding
locus datA promotes DnaA-ATP hydrolysis to enable
cell cycle-coordinated replication initiation. Proc.
Natl. Acad. Sci. U.S.A. 110, 936–941. http://dx.doi.
org/10.1073/pnas.1212070110
Katayama, T. (2001). Feedback controls restrain the
initiation of Escherichia coli chromosomal replication.
Mol. Microbiol. 41, 9–17.
Katayama, T., Kubota, T., Kurokawa, K., Crooke, E., and
Sekimizu, K. (1998). The initiator function of DnaA
protein is negatively regulated by the sliding clamp of the
E. coli chromosomal replicase. Cell 94, 61–71.
Katayama, T., Ozaki, S., Keyamura, K., and Fujimitsu, K.
(2010). Regulation of the replication cycle: conserved
and diverse regulatory systems for DnaA and oriC. Nat.
Rev. Microbiol. 8, 163–170. http://dx.doi.org/10.1038/
nrmicro2314
Kato, J., and Katayama, T. (2001). Hda, a novel DnaA-related
protein, regulates the replication cycle in Escherichia coli.
EMBO J. 20, 4253–4262. http://dx.doi.org/10.1093/
emboj/20.15.4253
Keyamura, K., Abe, Y., Higashi, M., Ueda, T., and Katayama,
T. (2009). DiaA dynamics are coupled with changes
in initial origin complexes leading to helicase loading.
J. Biol. Chem. 284, 25038–25050. http://dx.doi.
org/10.1074/jbc.M109.002717
Keyamura, K., Fujikawa, N., Ishida, T., Ozaki, S.,
Su’etsugu, M., Fujimitsu, K., Kagawa, W., Yokoyama,
S., Kurumizaka, H., and Katayama, T. (2007). The
interaction of DiaA and DnaA regulates the replication
cycle in E. coli by directly promoting ATP DnaA-specific
initiation complexes. Genes Dev. 21, 2083–2099.
http://dx.doi.org/10.1101/gad.1561207
Khare, V., and Eckert, K.A. (2002). The proofreading 3′ → 5′
exonuclease activity of DNA polymerases: a kinetic
barrier to translesion DNA synthesis. Mutat. Res. 510,
45–54.
Kiefer, J.R., Mao, C., Hansen, C.J., Basehore, S.L., Hogrefe,
H.H., Braman, J.C., and Beese, L.S. (1997). Crystal
structure of a thermostable Bacillus DNA polymerase I
large fragment at 2.1 A resolution. Structure 5, 95–108.
Kim, S.R., Matsui, K., Yamada, M., Gruz, P., and Nohmi,
T. (2001). Roles of chromosomal and episomal dinB
genes encoding DNA pol IV in targeted and untargeted
mutagenesis in Escherichia coli. Mol. Genet. Genomics
266, 207–215.
Kitagawa, R., Ozaki, T., Moriya, S., and Ogawa, T. (1998).
Negative control of replication initiation by a novel
chromosomal locus exhibiting exceptional affinity for
Escherichia coli DnaA protein. Genes Dev. 12, 3032–
3043.
Kobayashi, K., Ehrlich, S.D., Albertini, A., Amati, G.,
Andersen, K.K., Arnaud, M., Asai, K., Ashikaga, S.,
Aymerich, S., Bessieres, P., et al. (2003). Essential Bacillus
subtilis genes. Proc. Natl. Acad. Sci. U.S.A. 100, 4678–
4683. http://dx.doi.org/10.1073/pnas.0730515100
Kogoma, T. (1997). Stable DNA replication: interplay
between DNA replication, homologous recombination,
and transcription. Microbiol. Mol. Biol. Rev. 61,
212–238.
Köhler, P., and Marahiel, M.A. (1997). Association of the
histone-like protein HBsu with the nucleoid of Bacillus
subtilis. J. Bacteriol. 179, 2060–2064.
Kokoska, R.J., Bebenek, K., Boudsocq, F., Woodgate, R.,
and Kunkel, T.A. (2002). Low fidelity DNA synthesis
by a Y family DNA polymerase due to misalignment in
the active site. J. Biol. Chem. 277, 19633–19638. http://
dx.doi.org/10.1074/jbc.M202021200
Kornberg, A., and Baker, T.A. (1992a). Genome origins. In
DNA replication, 2nd edn, A. Kornberg, and T.A. Baker,
eds. 511–552.
Kornberg, A., and Baker, T.A. (1992b). Prokaryotic DNA
polymerases other than E. coli Pol I. In DNA replication,
2nd edn, A. Kornberg, and T.A. Baker, eds. 165–196.
Krause, M., Rückert, B., Lurz, R., and Messer, W. (1997).
Complexes at the replication origin of Bacillus subtilis
with homologous and heterologous DnaA protein. J.
Mol. Biol. 274, 365–380.
Kunkel, T.A., and Bebenek, K. (2000). DNA replication
fidelity. Annu. Rev. Biochem. 69, 497–529.
Kurz, M., Dalrymple, B., Wijffels, G., and Kongsuwan, K.
(2004). Interaction of the sliding clamp beta-subunit
and Hda, a DnaA-related protein. J. Bacteriol.

186, 3508–3515. http://dx.doi.org/10.1128/
JB.186.11.3508-3515.2004
Langston, L.D., Indiani, C., and O’Donnell, M. (2009).
Whither the replisome: emerging perspectives on the
dynamic nature of the DNA replication machinery. Cell
Cycle 8, 2686–2691.
Langston, L.D., and O’Donnell, M. (2006). DNA
replication: keep moving and don’t mind the gap.
Mol. Cell 23, 155–160. http://dx.doi.org/10.1016/j.
molcel.2006.05.034
Le Chatelier, E., Bécherel, O.J., d’Alençon, E., Canceill,
D., Ehrlich, S.D., Fuchs, R.P., and Jannière, L. (2004).
Involvement of DnaE, the second replicative DNA
polymerase from Bacillus subtilis, in DNA mutagenesis.
J. Biol. Chem. 279, 1757–1767. http://dx.doi.
org/10.1074/jbc.M310719200
Lecointe, F., Sérèna, C., Velten, M., Costes, A., McGovern,
S., Meile, J.C., Errington, J., Ehrlich, S.D., Noirot, P.,
and Polard, P. (2007). Anticipating chromosomal
replication fork arrest: SSB targets repair DNA helicases
to active forks. EMBO J. 26, 4239–4251. http://dx.doi.
org/10.1038/sj.emboj.7601848
Lee, P.S., Lin, D.C., Moriya, S., and Grossman, A.D. (2003).
Effects of the chromosome partitioning protein Spo0J
(ParB) on oriC positioning and replication initiation in
Bacillus subtilis. J. Bacteriol. 185, 1326–1337.
Lemon, K.P., and Grossman, A.D. (1998). Localization of
bacterial DNA polymerase: evidence for a factory model
of replication. Science 282, 1516–1519.
Lemon, K.P., and Grossman, A.D. (2000). Movement of
replicating DNA through a stationary replisome. Mol.
Cell 6, 1321–1330.
Lemon, K.P., Kurtser, I., Wu, J., and Grossman, A.D. (2000).
Control of initiation of sporulation by replication
initiation genes in Bacillus subtilis. J. Bacteriol. 182,
2989–2991.
Lemon, K.P., Moriya, S., Ogasawara, N., and Grossman,
A.D. (2002). Chromosome replication and segregation.
In Bacillus subtilis and its closest relatives: from genes to
cells, A.L. Sonenshein, J.A. Hoch, and R. Losick, eds.
73–86.
Lenne-Samuel, N., Wagner, J., Etienne, H., and Fuchs, R.P.
(2002). The processivity factor beta controls DNA
polymerase IV traffic during spontaneous mutagenesis
and translesion synthesis in vivo. EMBO Rep. 3, 45–49.
http://dx.doi.org/10.1093/embo-reports/kvf007
Leonard, T.A., Butler, P.J., and Löwe, J. (2005). Bacterial
chromosome segregation: structure and DNA binding
of the Soj dimer – a conserved biological switch.
EMBO J. 24, 270–282. http://dx.doi.org/10.1038/
sj.emboj.7600530
Lestini, R., and Michel, B. (2007). UvrD controls the access
of recombination proteins to blocked replication forks.
EMBO J. 26, 3804–3814. http://dx.doi.org/10.1038/
sj.emboj.7601804
Lindner, C., Nijland, R., van Hartskamp, M., Bron, S.,
Hamoen, L.W., and Kuipers, O.P. (2004). Differential
expression of two paralogous genes of Bacillus subtilis
encoding single-stranded DNA binding protein. J.
Bacteriol. 186, 1097–1105.
Lindow, J.C., Kuwano, M., Moriya, S., and Grossman,
A.D. (2002). Subcellular localization of the Bacillus
Construction and Maintenance of a Replication Fork | 29
subtilis structural maintenance of chromosomes (SMC)
protein. Mol. Microbiol. 46, 997–1009.
Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2001).
Crystal structure of a Y-family DNA polymerase in
action: a mechanism for error-prone and lesion-bypass
replication. Cell 107, 91–102.
Løbner-Olesen, A., Skovgaard, O., and Marinus, M.G.
(2005). Dam methylation: coordinating cellular
processes. Curr. Opin. Microbiol. 8, 154–160. http://
dx.doi.org/10.1016/j.mib.2005.02.009
López de Saro, F.J., Georgescu, R.E., Goodman, M.F., and
O’Donnell, M. (2003). Competitive processivity-clamp
usage by DNA polymerases during DNA replication
and repair. EMBO J. 22, 6408–6418. http://dx.doi.
org/10.1093/emboj/cdg603
Loscha, K.V., Jaudzems, K., Ioannou, C., Su, X.C., Hill, F.R.,
Otting, G., Dixon, N.E., and Liepinsh, E. (2009). A novel
zinc-binding fold in the helicase interaction domain of
the Bacillus subtilis DnaI helicase loader. Nucleic Acids
Res. 37, 2395–2404. http://dx.doi.org/10.1093/nar/
gkp092
Lu, M., Campbell, J.L., Boye, E., and Kleckner, N. (1994).
SeqA: a negative modulator of replication initiation in E.
coli. Cell 77, 413–426.
Maciag, M., Kochanowska, M., Lyzeń, R., Wegrzyn, G., and
Szalewska-Pałasz, A. (2010). ppGpp inhibits the activity
of Escherichia coli DnaG primase. Plasmid 63, 61–67.
http://dx.doi.org/10.1016/j.plasmid.2009.11.002
Maciąg, M., Nowicki, D., Janniere, L., Szalewska-Pałasz,
A., and Węgrzyn, G. (2011). Genetic response
to metabolic fluctuations: correlation between
central carbon metabolism and DNA replication in
Escherichia coli. Microb. Cell Fact. 10, 19. http://dx.doi.
org/10.1186/1475-2859-10-19
Makowska-Grzyska, M., and Kaguni, J.M. (2010).
Primase directs the release of DnaC from DnaB.
Mol. Cell 37, 90–101. http://dx.doi.org/10.1016/j.
molcel.2009.12.031
Maor-Shoshani, A., and Livneh, Z. (2002). Analysis of the
stimulation of DNA polymerase V of Escherichia coli by
processivity proteins. Biochemistry 41, 14438–14446.
Marczynski, G.T., and Shapiro, L. (2002). Control of
chromosome replication in Caulobacter crescentus.
Annu. Rev. Microbiol. 56, 625–656. http://dx.doi.
org/10.1146/annurev.micro.56.012302.161103
Marians, K.J. (2000). PriA-directed replication fork restart
in Escherichia coli. Trends Biochem. Sci. 25, 185–189.
Marians, K.J. (2004). Mechanisms of replication fork restart
in Escherichia coli. Phil. Trans. R. Soc. Lond. B Biol. Sci.
359, 71–77. http://dx.doi.org/10.1098/rstb.2003.1366
Marsin, S., McGovern, S., Ehrlich, S.D., Bruand, C.,
and Polard, P. (2001). Early steps of Bacillus subtilis
primosome assembly. J. Biol. Chem. 276, 45818–45825.
Marszalek, J., Zhang, W., Hupp, T.R., Margulies, C., Carr,
K.M., Cherry, S., and Kaguni, J.M. (1996). Domains of
DnaA protein involved in interaction with DnaB protein,
and in unwinding the Escherichia coli chromosomal
origin. J. Biol. Chem. 271, 18535–18542.
Martínez-Jiménez, M.I., Mesa, P., and Alonso, J.C. (2002).
Bacillus subtilis tau subunit of DNA polymerase III
interacts with bacteriophage SPP1 replicative DNA
helicase G40P. Nucleic Acids Res. 30, 5056–5064.
30 | Murray et al.
Masai, H., Deneke, J., Furui, Y., Tanaka, T., and Arai,
K.I. (1999). Escherichia coli and Bacillus subtilis PriA
proteins essential for recombination-dependent DNA
replication: involvement of ATPase/helicase activity of
PriA for inducible stable DNA replication. Biochimie
81, 847–857.
Mascarenhas, J., Sanchez, H., Tadesse, S., Kidane, D.,
Krisnamurthy, M., Alonso, J.C., and Graumann, P.L.
(2006). Bacillus subtilis SbcC protein plays an important
role in DNA inter-strand cross-link repair. BMC Mol.
Biol. 7, 20. http://dx.doi.org/10.1186/1471-2199-7-20
Mascarenhas, J., Soppa, J., Strunnikov, A.V., and Graumann,
P.L. (2002). Cell cycle-dependent localization of
two novel prokaryotic chromosome segregation and
condensation proteins in Bacillus subtilis that interact
with SMC protein. EMBO J. 21, 3108–3118.
McGlynn, P., and Lloyd, R.G. (2002). Recombinational
repair and restart of damaged replication forks. Nat. Rev.
Mol. Cell Biol. 3, 859–870. http://dx.doi.org/10.1038/
nrm951
McHenry, C.S. (2003). Chromosomal replicases as
asymmetric dimers: studies of subunit arrangement
and functional consequences. Mol. Microbiol. 49,
1157–1165.
McHenry, C.S. (2011). DNA replicases from a bacterial
perspective. Annu. Rev. Biochem. 80, 403–436. http://
dx.doi.org/10.1146/annurev-biochem-061208-091655
Meile, J.C., Wu, L.J., Ehrlich, S.D., Errington, J., and Noirot,
P. (2006). Systematic localisation of proteins fused
to the green fluorescent protein in Bacillus subtilis:
identification of new proteins at the DNA replication
factory. Proteomics 6, 2135–2146.
Merrikh, H., Machón, C., Grainger, W.H., Grossman,
A.D., and Soultanas, P. (2011). Co-directional
replication-transcription conflicts lead to replication
restart. Nature 470, 554–557. http://dx.doi.
org/10.1038/nature09758
Messer, W. (2002). The bacterial replication initiator DnaA.
DnaA and oriC, the bacterial mode to initiate DNA
replication. FEMS Microbiol. Rev. 26, 355–374.
Michel, B., Grompone, G., Florès, M.J., and Bidnenko, V.
(2004). Multiple pathways process stalled replication
forks. Proc. Natl. Acad. Sci. U.S.A. 101, 12783–12788.
http://dx.doi.org/10.1073/pnas.0401586101
Migocki, M.D., Lewis, P.J., Wake, R.G., and Harry, E.J.
(2004). The midcell replication factory in Bacillus
subtilis is highly mobile: implications for coordinating
chromosome replication with other cell cycle events.
Mol. Microbiol. 54, 452–463.
Mijakovic, I., Petranovic, D., Macek, B., Cepo, T., Mann,
M., Davies, J., Jensen, P.R., and Vujaklija, D. (2006).
Bacterial single-stranded DNA-binding proteins are
phosphorylated on tyrosine. Nucleic Acids Res. 34,
1588–1596. http://dx.doi.org/10.1093/nar/gkj514
Molle, V., Fujita, M., Jensen, S.T., Eichenberger, P.,
González-Pastor, J.E., Liu, J.S., and Losick, R. (2003).
The Spo0A regulon of Bacillus subtilis. Mol. Microbiol.
50, 1683–1701.
Moriya, S., Imai, Y., Hassan, A.K., and Ogasawara, N. (1999).
Regulation of initiation of Bacillus subtilis chromosome
replication. Plasmid 41, 17–29.
Moriya, S., Kato, K., Yoshikawa, H., and Ogasawara, N.
(1990). Isolation of a dnaA mutant of Bacillus subtilis
defective in initiation of replication: amount of DnaA
protein determines cells’ initiation potential. EMBO J.
9, 2905–2910.
Mott, M.L., and Berger, J.M. (2007). DNA replication
initiation: mechanisms and regulation in bacteria. Nat.
Rev. Microbiol. 5, 343–354. http://dx.doi.org/10.1038/
nrmicro1640
Mulcair, M.D., Schaeffer, P.M., Oakley, A.J., Cross, H.F.,
Neylon, C., Hill, T.M., and Dixon, N.E. (2006). A
molecular mousetrap determines polarity of termination
of DNA replication in E. coli. Cell 125, 1309–1319.
http://dx.doi.org/10.1016/j.cell.2006.04.040
Murray, H., and Errington, J. (2008). Dynamic control of
the DNA replication initiation protein DnaA by Soj/
ParA. Cell 135, 74–84. http://dx.doi.org/10.1016/j.
cell.2008.07.044
Murray, H., and Koh, A. (2014). Multiple regulatory
systems coordinate DNA replication with cell growth
in Bacillus subtilis. PLoS Genet. 10, e1004731. http://
dx.doi.org/10.1371/journal.pgen.1004731
Narula, J., Kuchina, A., Lee, D.Y., Fujita, M., Süel, G.M.,
and Igoshin, O.A. (2015). Chromosomal arrangement
of phosphorelay genes couples sporulation and
DNA replication. Cell 162, 328–337. http://dx.doi.
org/10.1016/j.cell.2015.06.012
Neylon, C., Kralicek, A.V., Hill, T.M., and Dixon, N.E.
(2005). Replication termination in Escherichia coli:
structure and antihelicase activity of the Tus-Ter
complex. Microbiol. Mol. Biol. Rev. 69, 501–526.
http://dx.doi.org/10.1128/MMBR.69.3.501-526.2005
Noirot-Gros, M.F., Dervyn, E., Wu, L.J., Mervelet, P.,
Errington, J., Ehrlich, S.D., and Noirot, P. (2002). An
expanded view of bacterial DNA replication. Proc.
Natl. Acad. Sci. U.S.A. 99, 8342–8347. http://dx.doi.
org/10.1073/pnas.122040799
Noirot-Gros, M.F., Velten, M., Yoshimura, M., McGovern,
S., Morimoto, T., Ehrlich, S.D., Ogasawara, N., Polard,
P., and Noirot, P. (2006). Functional dissection of YabA,
a negative regulator of DNA replication initiation in
Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 103, 2368–
2373. http://dx.doi.org/10.1073/pnas.0506914103
Nolivos, S., and Sherratt, D. (2014). The bacterial
chromosome: architecture and action of bacterial
SMC and SMC-like complexes. FEMS Microbiol.
Rev. 38, 380–392. http://dx.doi.org/10.1111/1574-
6976.12045
O’Donnell, M. (2006). Replisome architecture and
dynamics in Escherichia coli. J. Biol. Chem. 281, 10653–
10656. http://dx.doi.org/10.1074/jbc.R500028200
Oakley, A.J., Loscha, K.V., Schaeffer, P.M., Liepinsh, E.,
Pintacuda, G., Wilce, M.C., Otting, G., and Dixon,
N.E. (2005). Crystal and solution structures of the
helicase-binding domain of Escherichia coli primase.
J. Biol. Chem. 280, 11495–11504. http://dx.doi.
org/10.1074/jbc.M412645200
Ogasawara, N., Moriya, S., Mazza, G., and Yoshikawa, H.
(1986). A Bacillus subtilis dnaG mutant harbours a
mutation in a gene homologous to the dnaN gene of
Escherichia coli. Gene 45, 227–231.
Ogawa, T., Yamada, Y., Kuroda, T., Kishi, T., and Moriya,
S. (2002). The datA locus predominantly contributes
to the initiator titration mechanism in the control of

replication initiation in Escherichia coli. Mol. Microbiol.
44, 1367–1375.
Ogura, Y., Ogasawara, N., Harry, E.J., and Moriya, S. (2003).
Increasing the ratio of Soj to Spo0J promotes replication
initiation in Bacillus subtilis. J. Bacteriol. 185, 6316–6324.
Ohmori, H., Friedberg, E.C., Fuchs, R.P., Goodman, M.F.,
Hanaoka, F., Hinkle, D., Kunkel, T.A., Lawrence, C.W.,
Livneh, Z., Nohmi, T., et al. (2001). The Y-family of
DNA polymerases. Mol. Cell 8, 7–8.
Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., and
Sugino, A. (1968). Mechanism of DNA chain growth. I.
Possible discontinuity and unusual secondary structure
of newly synthesized chains. Proc. Natl. Acad. Sci. U.S.A.
59, 598–605.
Okumura, H., Yoshimura, M., Ueki, M., Oshima, T.,
Ogasawara, N., and Ishikawa, S. (2012). Regulation of
chromosomal replication initiation by oriC-proximal
DnaA-box clusters in Bacillus subtilis. Nucleic Acids Res.
40, 220–234. http://dx.doi.org/10.1093/nar/gkr716
Orr, E., and Staudenbauer, W.L. (1982). Bacillus subtilis
DNA gyrase: purification of subunits and reconstitution
of supercoiling activity. J. Bacteriol. 151, 524–527.
Petit, M.A., Dervyn, E., Rose, M., Entian, K.D., McGovern,
S., Ehrlich, S.D., and Bruand, C. (1998). PcrA is an
essential DNA helicase of Bacillus subtilis fulfilling
functions both in repair and rolling-circle replication.
Mol. Microbiol. 29, 261–273.
Petit, M.A., and Ehrlich, D. (2002). Essential bacterial
helicases that counteract the toxicity of recombination
proteins. EMBO J. 21, 3137–3147. http://dx.doi.
org/10.1093/emboj/cdf317
Petit, M.A., and Ehrlich, S.D. (2000). The NAD-dependent
ligase encoded by yerG is an essential gene of Bacillus
subtilis. Nucleic Acids Res. 28, 4642–4648.
Pham, P., Bertram, J.G., O’Donnell, M., Woodgate, R., and
Goodman, M.F. (2001). A model for SOS-lesion-targeted
mutations in Escherichia coli. Nature 409, 366–370.
http://dx.doi.org/10.1038/35053116
Pham, P., Seitz, E.M., Saveliev, S., Shen, X., Woodgate, R.,
Cox, M.M., and Goodman, M.F. (2002). Two distinct
modes of RecA action are required for DNA polymerase
V-catalyzed translesion synthesis. Proc. Natl. Acad. Sci.
U.S.A. 99, 11061–11066. http://dx.doi.org/10.1073/
pnas.172197099
Piggot, P.J., and Hilbert, D.W. (2004). Sporulation of
Bacillus subtilis. Curr. Opin. Microbiol. 7, 579–586.
http://dx.doi.org/10.1016/j.mib.2004.10.001
Polard, P., Marsin, S., McGovern, S., Velten, M., Wigley,
D.B., Ehrlich, S.D., and Bruand, C. (2002). Restart of
DNA replication in Gram-positive bacteria: functional
characterisation of the Bacillus subtilis PriA initiator.
Nucleic Acids Res. 30, 1593–1605.
Pomerantz, R.T., and O’Donnell, M. (2008). The replisome
uses mRNA as a primer after colliding with RNA
polymerase. Nature 456, 762–766. http://dx.doi.
org/10.1038/nature07527
Pomerantz, R.T., and O’Donnell, M. (2010). Direct
restart of a replication fork stalled by a head-on RNA
polymerase. Science 327, 590–592.
Potrykus, K., and Cashel, M. (2008). (p)ppGpp: still
magical? Annu. Rev. Microbiol. 62, 35–51. http://
dx.doi.org/10.1146/annurev.micro.62.081307.162903
Construction and Maintenance of a Replication Fork | 31
Prakash, S., Johnson, R.E., and Prakash, L. (2005).
Eukaryotic translesion synthesis DNA polymerases:
specificity of structure and function. Annu. Rev.
Biochem. 74, 317–353.
Raghunathan, S., Kozlov, A.G., Lohman, T.M., and
Waksman, G. (2000). Structure of the DNA binding
domain of E. coli SSB bound to ssDNA. Nat. Struct. Biol.
7, 648–652. http://dx.doi.org/10.1038/77943
Rahn-Lee, L., Gorbatyuk, B., Skovgaard, O., and Losick,
R. (2009). The conserved sporulation protein YneE
inhibits DNA replication in Bacillus subtilis. J. Bacteriol.
191, 3736–3739. http://dx.doi.org/10.1128/JB.00216-
09
Rahn-Lee, L., Merrikh, H., Grossman, A.D., and Losick, R.
(2011). The sporulation protein SirA inhibits the binding
of DnaA to the origin of replication by contacting a patch
of clustered amino acids. J. Bacteriol. 193, 1302–1307.
http://dx.doi.org/10.1128/JB.01390-10
Reuven, N.B., Arad, G., Maor-Shoshani, A., and Livneh,
Z. (1999). The mutagenesis protein UmuC is a DNA
polymerase activated by UmuD’, RecA, and SSB and
is specialized for translesion replication. J. Biol. Chem.
274, 31763–31766.
Richardson, T.T., Harran, O., and Murray, H. (2016). The
bacterial DnaA-trio replication origin element specifies
single-stranded DNA initiator binding. Nature 534,
412–416.
Rivas-Castillo, A.M., Yasbin, R.E., Robleto, E., Nicholson,
W.L., and Pedraza-Reyes, M. (2010). Role of the
Y-family DNA polymerases YqjH and YqjW in
protecting sporulating Bacillus subtilis cells from DNA
damage. Curr. Microbiol. 60, 263–267. http://dx.doi.
org/10.1007/s00284-009-9535-3
Robinson, A., and van Oijen, A.M. (2013). Bacterial
replication, transcription and translation: mechanistic
insights from single-molecule biochemical studies.
Nature Rev. Microbiol. 11, 303–315.
Rokop, M.E., Auchtung, J.M., and Grossman, A.D. (2004).
Control of DNA replication initiation by recruitment
of an essential initiation protein to the membrane of
Bacillus subtilis. Mol. Microbiol. 52, 1757–1767. http://
dx.doi.org/10.1111/j.1365-2958.2004.04091.x
Rowland, S.L., Burkholder, W.F., Cunningham, K.A.,
Maciejewski, M.W., Grossman, A.D., and King, G.F.
(2004). Structure and mechanism of action of Sda, an
inhibitor of the histidine kinases that regulate initiation
of sporulation in Bacillus subtilis. Mol. Cell 13, 689–701.
Ruvolo, M.V., Mach, K.E., and Burkholder, W.F. (2006).
Proteolysis of the replication checkpoint protein Sda
is necessary for the efficient initiation of sporulation
after transient replication stress in Bacillus subtilis. Mol.
Microbiol. 60, 1490–1508.
Rymer, R.U., Solorio, F.A., Tehranchi, A.K., Chu, C., Corn,
J.E., Keck, J.L., Wang, J.D., and Berger, J.M. (2012).
Binding mechanism of metal⋅NTP substrates and
stringent-response alarmones to bacterial DnaG-type
primases. Structure 20, 1478–1489. http://dx.doi.
org/10.1016/j.str.2012.05.017
Sagher, D., Turkington, E., Acharya, S., and Strauss,
B. (1994). Production of UV-induced frameshift
mutations in vitro by DNA polymerases deficient in
3′ → 5′ exonuclease activity. J. Mol. Biol. 240, 226–242.
32 | Murray et al.
Sanders, G.M., Dallmann, H.G., and McHenry, C.S.
(2010). Reconstitution of the B. subtilis replisome
with 13 proteins including two distinct replicases.
Mol. Cell 37, 273–281. http://dx.doi.org/10.1016/j.
molcel.2009.12.025
Sandler, S.J. (2000). Multiple genetic pathways for restarting
DNA replication forks in Escherichia coli K-12. Genetics
155, 487–497.
Sandler, S.J., and Marians, K.J. (2000). Role of PriA
in replication fork reactivation in Escherichia coli. J.
Bacteriol. 182, 9–13.
Sandler, S.J., Samra, H.S., and Clark, A.J. (1996). Differential
suppression of priA2::kan phenotypes in Escherichia coli
K-12 by mutations in priA, lexA, and dnaC. Genetics
143, 5–13.
Sanjanwala, B., and Ganesan, A.T. (1989). DNA polymerase
III gene of Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A.
86, 4421–4424.
Sanjanwala, B., and Ganesan, A.T. (1991). Genetic structure
and domains of DNA polymerase III of Bacillus subtilis.
Mol. Gen. Genet. 226, 467–472.
Scheuermann, R.H., and Echols, H. (1984). A separate
editing exonuclease for DNA replication: the epsilon
subunit of Escherichia coli DNA polymerase III
holoenzyme. Proc. Natl. Acad. Sci. U.S.A. 81, 7747–
7751.
Scholefield, G., Errington, J., and Murray, H. (2012).
Soj/ParA stalls DNA replication by inhibiting helix
formation of the initiator protein DnaA. EMBO J. 31,
1542–1555. http://dx.doi.org/10.1038/emboj.2012.6
Scholefield, G., and Murray, H. (2013). YabA and DnaD
inhibit helix assembly of the DNA replication initiation
protein DnaA. Mol. Microbiol. 90, 147–159. http://
dx.doi.org/10.1111/mmi.12353
Scholefield, G., Whiting, R., Errington, J., and Murray, H.
(2011). Spo0J regulates the oligomeric state of Soj to
trigger its switch from an activator to an inhibitor of DNA
replication initiation. Mol. Microbiol. 79, 1089–1100.
http://dx.doi.org/10.1111/j.1365-2958.2010.07507.x
Schvartzman, J.B., and Stasiak, A. (2004). A topological
view of the replicon. EMBO Rep. 5, 256–261. http://
dx.doi.org/10.1038/sj.embor.7400101
Sekimizu, K., and Kornberg, A. (1988). Cardiolipin
activation of dnaA protein, the initiation protein
of replication in Escherichia coli. J. Biol. Chem. 263,
7131–7135.
Séror, S.J., Levine, A., and Vannier, F. (1991). Post-initiation
control of chromosomal replication in Bacillus subtilis:
a mechanism for limiting over-replication or for
duplicating key growth and sporulation genes? Res.
Microbiol. 142, 861–867.
Shereda, R.D., Bernstein, D.A., and Keck, J.L. (2007).
A central role for SSB in Escherichia coli RecQ DNA
helicase function. J. Biol. Chem. 282, 19247–19258.
http://dx.doi.org/10.1074/jbc.M608011200
Shereda, R.D., Reiter, N.J., Butcher, S.E., and Keck, J.L.
(2009). Identification of the SSB binding site on E. coli
RecQ reveals a conserved surface for binding SSB’s C
terminus. J. Mol. Biol. 386, 612–625. http://dx.doi.
org/10.1016/j.jmb.2008.12.065
Simmons, L.A., Davies, B.W., Grossman, A.D., and Walker,
G.C. (2008). Beta clamp directs localization of mismatch
repair in Bacillus subtilis. Mol. Cell 29, 291–301. http://
dx.doi.org/10.1016/j.molcel.2007.10.036
Smits, W.K., Goranov, A.I., and Grossman, A.D. (2010).
Ordered association of helicase loader proteins with
the Bacillus subtilis origin of replication in vivo. Mol.
Microbiol. 75, 452–461. http://dx.doi.org/10.1111/
j.1365-2958.2009.06999.x
Smits, W.K., Merrikh, H., Bonilla, C.Y., and Grossman,
A.D. (2011). Primosomal proteins DnaD and DnaB
are recruited to chromosomal regions bound by DnaA
in Bacillus subtilis. J. Bacteriol. 193, 640–648. http://
dx.doi.org/10.1128/JB.01253-10
Soppa, J., Kobayashi, K., Noirot-Gros, M.F., Oesterhelt, D.,
Ehrlich, S.D., Dervyn, E., Ogasawara, N., and Moriya, S.
(2002). Discovery of two novel families of proteins that
are proposed to interact with prokaryotic SMC proteins,
and characterization of the Bacillus subtilis family
members ScpA and ScpB. Mol. Microbiol. 45, 59–71.
Soufo, C.D., Soufo, H.J., Noirot-Gros, M.F., Steindorf,
A., Noirot, P., and Graumann, P.L. (2008).
Cell-cycle-dependent spatial sequestration of the
DnaA replication initiator protein in Bacillus subtilis.
Dev. Cell 15, 935–941. http://dx.doi.org/10.1016/j.
devcel.2008.09.010
Soultanas, P. (2005). The bacterial helicase-primase
interaction: a common structural/functional module.
Structure 13, 839–844. http://dx.doi.org/10.1016/j.
str.2005.04.006
Su’etsugu, M., Shimuta, T.R., Ishida, T., Kawakami, H., and
Katayama, T. (2005). Protein associations in DnaA-ATP
hydrolysis mediated by the Hda-replicase clamp
complex. J. Biol. Chem. 280, 6528–6536. http://dx.doi.
org/10.1074/jbc.M412060200
Su’etsugu, M., Takata, M., Kubota, T., Matsuda, Y., and
Katayama, T. (2004). Molecular mechanism of DNA
replication-coupled inactivation of the initiator protein
in Escherichia coli: interaction of DnaA with the sliding
clamp-loaded DNA and the sliding clamp-Hda complex.
Genes Cells 9, 509–522.
Sung, H.M., Yeamans, G., Ross, C.A., and Yasbin, R.E.
(2003). Roles of YqjH and YqjW, homologs of the
Escherichia coli UmuC/DinB or Y superfamily of DNA
polymerases, in stationary-phase mutagenesis and
UV-induced mutagenesis of Bacillus subtilis. J. Bacteriol.
185, 2153–2160.
Sutton, M.D., Carr, K.M., Vicente, M., and Kaguni, J.M.
(1998). Escherichia coli DnaA protein. The N-terminal
domain and loading of DnaB helicase at the E. coli
chromosomal origin. J. Biol. Chem. 273, 34255–34262.
Sweasy, J.B., Witkin, E.M., Sinha, N., and
Roegner-Maniscalco, V. (1990). RecA protein of
Escherichia coli has a third essential role in SOS mutator
activity. J. Bacteriol. 172, 3030–3036.
Syson, K., Thirlway, J., Hounslow, A.M., Soultanas, P.,
and Waltho, J.P. (2005). Solution structure of the
helicase-interaction domain of the primase DnaG: a
model for helicase activation. Structure 13, 609–616.
http://dx.doi.org/10.1016/j.str.2005.01.022
Taghbalout, A., Landoulsi, A., Kern, R., Yamazoe, M.,
Hiraga, S., Holland, B., Kohiyama, M., and Malki, A.
(2000). Competition between the replication initiator
DnaA and the sequestration factor SeqA for binding to

the hemimethylated chromosomal origin of E. coli in
vitro. Genes Cells 5, 873–884.
Tamanoi, F., Okazaki, T., and Okazaki, R. (1977). Persistence
of RNA attached to nascent short DNA pieces in Bacillus
subtilis cells defective in DNA polymerase I. Biochem.
Biophys. Res. Commun. 77, 290–297.
Tang, M., Pham, P., Shen, X., Taylor, J.S., O’Donnell, M.,
Woodgate, R., and Goodman, M.F. (2000). Roles of E.
coli DNA polymerases IV and V in lesion-targeted and
untargeted SOS mutagenesis. Nature 404, 1014–1018.
Thirlway, J., and Soultanas, P. (2006). In the Bacillus
stearothermophilus DnaB-DnaG complex, the activities
of the two proteins are modulated by distinct but
overlapping networks of residues. J. Bacteriol. 188,
1534–1539. http://dx.doi.org/10.1128/JB.188.4.1534-
1539.2006
Thirlway, J., Turner, I.J., Gibson, C.T., Gardiner, L., Brady,
K., Allen, S., Roberts, C.J., and Soultanas, P. (2004).
DnaG interacts with a linker region that joins the N- and
C-domains of DnaB and induces the formation of 3-fold
symmetric rings. Nucleic Acids Res. 32, 2977–2986.
http://dx.doi.org/10.1093/nar/gkh628
Thony, B., Hwang, D.S., Fradkin, L., and Kornberg, A.
(1991). iciA, an Escherichia coli gene encoding a specific
inhibitor of chromosomal initiation of replication in
vitro. Proc. Natl. Acad. Sci. U.S.A. 88, 4066–4070.
Tippin, B., Pham, P., and Goodman, M.F. (2004).
Error-prone replication for better or worse. Trends
Microbiol. 12, 288–295. http://dx.doi.org/10.1016/j.
tim.2004.04.004
Tougu, K., Peng, H., and Marians, K.J. (1994). Identification
of a domain of Escherichia coli primase required for
functional interaction with the DnaB helicase at the
replication fork. J. Biol. Chem. 269, 4675–4682.
Tsai, K.L., Lo, Y.H., Sun, Y.J., and Hsiao, C.D. (2009).
Molecular interplay between the replicative helicase
DnaC and its loader protein DnaI from Geobacillus
kaustophilus. J. Mol. Biol. 393, 1056–1069. http://
dx.doi.org/10.1016/j.jmb.2009.09.002
Turner, I.J., Scott, D.J., Allen, S., Roberts, C.J., and Soultanas,
P. (2004). The Bacillus subtilis DnaD protein: a putative
link between DNA remodeling and initiation of DNA
replication. FEBS Lett. 577, 460–464. http://dx.doi.
org/10.1016/j.febslet.2004.10.051
Veening, J.W., Murray, H., and Errington, J. (2009). A
mechanism for cell cycle regulation of sporulation
initiation in Bacillus subtilis. Genes Dev. 23, 1959–1970.
http://dx.doi.org/10.1101/gad.528209
Velten, M., McGovern, S., Marsin, S., Ehrlich, S.D., Noirot,
P., and Polard, P. (2003). A two-protein strategy for the
functional loading of a cellular replicative DNA helicase.
Mol. Cell 11, 1009–1020.
von Freiesleben, U., Rasmussen, K.V., and Schaechter, M.
(1994). SeqA limits DnaA activity in replication from
oriC in Escherichia coli. Mol. Microbiol. 14, 763–772.
Wagner, J., Etienne, H., Janel-Bintz, R., and Fuchs, R.P.
(2002). Genetics of mutagenesis in E. coli: various
Construction and Maintenance of a Replication Fork | 33
combinations of translesion polymerases (Pol II, IV and
V) deal with lesion/sequence context diversity. DNA
Repair. 1, 159–167.
Wagner, J., Gruz, P., Kim, S.R., Yamada, M., Matsui, K.,
Fuchs, R.P., and Nohmi, T. (1999). The dinB gene
encodes a novel E. coli DNA polymerase, DNA pol IV,
involved in mutagenesis. Mol. Cell 4, 281–286.
Wagner, J., and Nohmi, T. (2000). Escherichia coli DNA
polymerase IV mutator activity: genetic requirements
and mutational specificity. J. Bacteriol. 182, 4587–4595.
Wagner, J.K., Marquis, K.A., and Rudner, D.Z. (2009). SirA
enforces diploidy by inhibiting the replication initiator
DnaA during spore formation in Bacillus subtilis. Mol.
Microbiol. 73, 963–974.
Wang, J.D., Sanders, G.M., and Grossman, A.D. (2007).
Nutritional control of elongation of DNA replication
by (p)ppGpp. Cell 128, 865–875. http://dx.doi.
org/10.1016/j.cell.2006.12.043
Weller, G.R., Kysela, B., Roy, R., Tonkin, L.M., Scanlan,
E., Della, M., Devine, S.K., Day, J.P., Wilkinson, A.,
d’Adda di Fagagna, F., et al. (2002). Identification of a
DNA nonhomologous end-joining complex in bacteria.
Science 297, 1686–1689. http://dx.doi.org/10.1126/
science.1074584
Wijffels, G., Dalrymple, B., Kongsuwan, K., and
Dixon, N.E. (2005). Conservation of eubacterial
replicases. IUBMB Life 57, 413–419. http://dx.doi.
org/10.1080/15216540500138246
Wijffels, G., Dalrymple, B.P., Prosselkov, P., Kongsuwan,
K., Epa, V.C., Lilley, P.E., Jergic, S., Buchardt, J., Brown,
S.E., Alewood, P.F., et al. (2004). Inhibition of protein
interactions with the beta 2 sliding clamp of Escherichia
coli DNA polymerase III by peptides from beta 2-binding
proteins. Biochemistry 43, 5661–5671. http://dx.doi.
org/10.1021/bi036229j
Xia, W., and Dowhan, W. (1995). In vivo evidence for the
involvement of anionic phospholipids in initiation of
DNA replication in Escherichia coli. Proc. Natl. Acad. Sci.
U.S.A. 92, 783–787.
Xu, L., and Marians, K.J. (2003). PriA mediates DNA
replication pathway choice at recombination
intermediates. Mol. Cell 11, 817–826.
Yasuda, H., and Okazaki, T. (1985). Alkali-labile structures
linked to the 5′ ends of Bacillus subtilis short DNA chains.
Nucleic Acids Res. 13, 4953–4969.
Zhang, W., Carneiro, M.J., Turner, I.J., Allen, S., Roberts,
C.J., and Soultanas, P. (2005). The Bacillus subtilis DnaD
and DnaB proteins exhibit different DNA remodelling
activities. J. Mol. Biol. 351, 66–75. http://dx.doi.
org/10.1016/j.jmb.2005.05.065
Zhang, W., Machón, C., Orta, A., Phillips, N., Roberts, C.J.,
Allen, S., and Soultanas, P. (2008). Single-molecule
atomic force spectroscopy reveals that DnaD forms
scaffolds and enhances duplex melting. J. Mol.
Biol. 377, 706–714. http://dx.doi.org/10.1016/j.
jmb.2008.01.067
Dynamics of DNA Double-strand Break
Repair in Bacillus subtilis
Begoña Carrasco, Paula P. Cárdenas, Ester Serrano, Rubén Torres,
Elena M. Seco, Silvia Ayora and Juan C. Alonso*
Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CNB-CSIC, Campus Universidad
Autónoma de Madrid, Madrid, Spain.
*Correspondence: jcalonso@cnb.csic.es
https://doi.org/10.21775/9781910190579-02
Abstract
All organisms have developed a variety of DNA
repair mechanisms to cope with DNA double
strands breaks (DSBs). In replicating cells, homolo-
gous recombination (HR), which uses an intact
homologous template to restore lost information
at the break site, is the main pathway for error-free
repair of one- or two-ended DSBs and for pro-
moting the re-establishment of replication forks
during vegetative growth. Genetic, cytological
and biochemical approaches were used to analyse
the requirements of exponentially growing Bacil-
lus subtilis cells to survive broken DNA ends and
to visualize the choreography of DSB repair. The
damage-induced multi-protein complex (recombi-
nosome), organized into focal assemblies, has been
confirmed by biochemical approaches. HR is coor-
dinated with other essential processes, such as DNA
replication, transcription and chromosomal segre-
gation. When DSB recognition or end resection are
severely impaired or when an intact homologous
template is simply not available, the ends should
be reconnected by template-independent mecha-
nisms using minimal (single-strand annealing) or
no-sequence-homology (non-homologous end
joining). These effective repair mechanisms lead
to loss of genetic information (if the DNA ends
are altered before re-ligation), and to chromosomal
rearrangements (if incorrect ends are rejoined).
2
Introduction
The faithful replication and the maintenance of the
genome(s) are of primary importance for all living
organisms. In response to DNA damage, cells alter
their chromatin structure (nucleoid in bacteria)
and utilize dedicated error-free pathways [e.g. base
excision repair (BER), nucleotide excision repair
(NER), mismatch repair (MMR), etc.] to remove
DNA lesions and misincorporated nucleotides.
Persistent DNA damage can induce an intricate
series of interlocking signalling reactions, known
as the DNA damage response (DDR). The DDR
coordinates cell cycle arrest (inhibition of DNA
replication in bacteria), activates transcriptional
programs and initiates DNA repair (Zhou and
Elledge, 2000; Friedberg et al., 2006; Cimprich and
Cortez, 2008; Ciccia and Elledge, 2010; Jackson
and Bartek, 2009; Ditch and Paull, 2012; Chapman
et al., 2012). This DDR, which plays an essential
role in ensuring rapid detection of DNA damage
and in selecting the repair pathway needed to repair
the damage, can be divided into a series of distinct,
but perhaps functionally interrelated, pathways
defined by the type of intermediates accumulated:
single strand [ss] DNA regions, 3′-tailed duplex or
Holliday junction (HJ) intermediates (Zhou and
Elledge, 2000; Friedberg et al., 2006, Cimprich and
Cortez, 2008; Ciccia and Elledge, 2010; Jackson
and Bartek, 2009).
36 | Carrasco et al.
The genomes of most bacterial species consist of
one circular chromosome that is several million base
pairs in length (Luijsterburg et al., 2008). In Bacillus
subtilis the circular genome is » 2 mm in circumfer-
ence, has a volume of » 200 µm3 and is compacted
into » 0.5 µm3, with a total cell volume going from
2.5 to 4.5 fl during exponential growth (Yu et al.,
2014). The overall shape of the bacterial nucleoid
appears as a compressed helical structure, and mul-
tiple factors cooperate to compact the chromosome,
such as small nucleoid-binding proteins (NAPs,
Histone-like, e.g. HBsu, Lrp), structural mainte-
nance of chromosomes proteins (SMC-ScpAB),
enzymes that affect DNA supercoiling (type I and
II topoisomerases), DNA segregation (Soj-Spo0J)
proteins, and RNA molecules. In bacteria, DNA
replication, transcription and segregation occur
concurrently, with the coupling of transcription and
translation and DNA replication as the main forces
that expand the nucleoid. Daughter chromosome
regions are moved away from each other as replica-
tion proceeds (see Chapters 1 and 3). The degree of
nucleoid compaction varies as a function of the cel-
lular growth phase and rate. The dynamic structure
of the genome, which is composed of numerous
supercoiled loops (Postow et al., 2004), is essential
for life. The SMC–ScpAB complexes are recruited
to the oriC region by specific contacts with Spo0J
(also termed ParB) bound to parS sites (Gruber
and Errington, 2009, Sullivan et al., 2009), but
subsequently shift away from parS sites to promote
long-range contacts along the entire replichores
(Marbouty et al., 2015, Kim and Loparo, 2016).
These proteins also facilitate the diffusion of the
broken DNA end to find an intact sister strand or
chromosome through condensation/decondensa-
tion and re-organization of the nucleoids, and help
to maintain the torsional tension in the DNA, con-
tributing to supercoiling which is mainly achieved
by the action of DNA topoisomerases (reviewed
by Sherratt, 2003; Frenkiel-Krispin and Minsky,
2006; Witz and Stasiak, 2010). Using a yeast two-
hybrid system it was shown that ScpA interacts
with AddB, a nuclease subunit of the AddAB heli-
case–nucleases complex (counterpart of Escherichia
coli RecBCD or Mycobacteria tuberculosis AdnAB)
(Dervyn et al., 2004), which acts early in HR. The
lack of the SMC–ScpAB complex affects many
DNA metabolic processes, e.i. DNA replication,
and chromosome segregation in B. subtilis cells, but
the role of ScpA or the SMC–ScpAB complex in
DSB repair is poorly understood.
The absence of many of the known DNA repair
proteins, in an otherwise wild type (wt) background,
does not show a synthetic lethality. However, some
mutations in genes involved in DNA recombination
result in an accumulation of DNA recombination
intermediates that may be toxic for growth. For
example, in the absence of the recombinase loaders
(e.g. RecO, RecR, RecF) mutations in pcrA gene
become viable. Furthermore, the absence of both
branch migration translocases (RecG and RuvAB)
or of proteins processing double Holliday junctions
(HJ) (RuvAB and RecU (counterpart of E. coli
RuvC) render cells synthetically lethal (Fig. 2.1 and
Table 2.1) (see Carrasco et al., 2012). In contrast,
in E. coli, the corresponding double mutant cells
are viable (Kuzminov, 1999, McGlynn and Lloyd,
2002; Courcelle and Hanawalt, 2003; Kreuzer,
2005; Persky and Lovett, 2008; Michel et al., 2007).
During exponential growth in rich medium and
at 37°C, B. subtilis cells divide every 24–28 min in
Luria–Bertani (LB) medium, while their shortest
chromosome replication time is ~40 min, suggest-
ing that cells might have either four or even eight
replication origins per cell leading to ‘multifork rep-
lication’ (Quinn and Sueoka, 1970; Lemon et al.,
Figure 2.1 Schematic grouping of B. subtilis
RecA-dependent DNA recombinational repair genes
into different epistatic groups. Since a recA mutation
is epistatic with any representative mutation on the
different epistatic groups (α to κ), it is placed in the
centre.
Another random document with
no related content on Scribd:
imperialism. But he is also given a glimpse of a people who retain a
regard for the rights and dignity of the individual, a people who have
turned away from violence and shown a remarkable capacity for
achieving change without sacrificing stability, for combining growth
with order.

THE UNITED STATES: A SELF-PORTRAIT

De Forest, in Playing the Mischief, describes Congressman Sykes
Drummond as a “Robert-the-Devil” type. This complete cynic makes
an interesting comment on the conditions around him: “A John Bull
told me yesterday that there is no such thing known in England as a
municipal ring or a thieving mayor. That is what any American of the
present day would set down as a fairy story.” If the political novel has
any validity as a commentary on national characteristics, one
conclusion is inescapable: many Americans become criminals when
they accept public office. Drummond’s comment is borne out by the
two groups of novels. In the English novel individuals such as Hamer
Shawcross and Chester Nimmo sell out to the opposition. Nimmo
even engages in commercial activities too closely related to his
official duties to be quite proper. But there is a complete absence of
the corruption portrayed at all levels of government in the American
political novel.
Crawford attempted to differentiate the English from the American
early in An American Politician. The novel’s political naïveté renders
any of its judgments suspect, but in this Crawford appears to come
close to the truth. His opinion is that
English people ... love to associate with persons of rank and power from a
disinterested love of these things themselves, whereas in most other
countries the society of notable and influential persons is chiefly sought
from the most cynical motives of personal advantage.... But politics in
England and politics in America, so far as the main points are concerned,
are as different as it is possible for any two social functions to be. Roughly,
Government and the doings of Government are centripetal in England,
and centrifugal in America. In England the will of the people assists the
working of Providence, whereas in America, devout persons pray that
 Providence may on occasion modify the will of the people. In England men
believe in the Queen, the Royal Family, the Established Church, and
Belgravia first, and in themselves afterwards. Americans believe in
themselves devoutly, and a man who could “establish” upon them a
church, a royalty, or a peerage, would be a very clever fellow.
His diagnosis of cynicism and self-interest as leading American
characteristics is echoed in the other novels. Good politicians do
appear, but for every Lincoln there are ten Boss Tweeds. Henry
Adams’ Democracy also contains passages in which American
political life is compared unfavorably with that of England. Madeleine
Lee attends an immensely boring White House reception. Not only
are the guests dull, but to her the President and his wife appear as
automatons aping royalty.

Forces of Corruption
Complete responsibility for corruption does not always rest with the
politician. Some office holders, like Honest John Vane, sincerely try
to stay clean. The corrupt politician usually has a collaborator in the
person of the man who buys him. In Garland’s A Spoil of Office
Bradley Talcott’s illusions are shattered in the legislature and in
Congress. Looking around him he finds that “to rob the
commonwealth was a joke.” State legislatures are often described as
assemblages of brigands. The “Woodchuck Session” of the
legislature in Coniston is arrant banditry, and the ones in The Plum
Tree and Mr. Crewe’s Career are only a little less obvious. The
industrialist is usually co-villain with his politician hireling. When he
does not appear in person, he is represented by his middleman, the
professional lobbyist. The lobbyist’s unscrupulous use of his trusting
wife is the subject of Frances Hodgson Burnett’s Through One
Administration (1914). Jacob Pike, in Playing the Mischief, regards
the institution with pride:
From his point of view, it was a kind of public life; it was more completely
“inside politics” than even electioneering or legislation; it was, as he
believed, the very germ and main-spring of statesmanship. A leading
lobbyist knew exactly how the world is governed, and for what purpose....
This whole aspect of the American governmental process in the
novel is a very unsavory one. The industrialists are predatory robber
barons who purchase dishonest politicians in order to obtain special
privilege. In The Charterhouse of Parma the Contessa dissuades
Fabrizio from going to America by explaining to him “the cult of the
god Dollar.” In several of his novels Sinclair declares that American
foreign policy has been determined by the industrial interests. He
portrays Dollar Diplomacy with a vengeance. In Oil! he contends that
the United States joined with Britain and France to fight the
Bolsheviks not because of political ideology, but because “the
creditor nations meant to make an example of Soviet Russia, and
establish the rule that a government which repudiated its debts
would be put out of business.” He also tells the reader that the same
oil interests which backed Harding had turned in and out of office a
succession of Mexican governments to suit their own commercial
purposes. And it is not hard to see in Nostromo the influence of the
American tycoon Holroyd at work when the American cruiser
Powhatan appears to salute the Occidental flag and “put an end to
the Costaguana-Sulaco War.”
If the perverters of power are not demagogues with messianic
complexes, they may be gangsters who rule by ballots when bribery
fails. These subjects in the novel did not go out of vogue with the
Muckraking Era or with Prohibition. A new rash of novels about
gangsters in politics has appeared in recent years. The gunman who
is concerned about investing his money may be replacing the one
who writes his name with machine gun bullets, but his influence in
politics is the same.

The American Idealist

The reverse side of this particular coin shows the idealist at work. He
may come out of the political wringer with his ideals mangled and his
illusions full of holes, but still he retains something of the impulse
which created the Declaration of Independence and the Constitution.
Millard Carroll in The Grand Design is such a one, but he emerges
as an old man at the end of his ordeal. Nick Burr in Ellen Glasgow’s
The Voice of the People (1900) is another, but his end is violent
death. In recent years this idealistic aspect of the American political
character has often found expression in heroes who engage in the
direct struggle for liberty. Both Robert Jordan and Glenn Spotswood
spill their blood on Spanish earth fighting against Franco. Were it not
for the crusading district attorneys of books such as Ashes and
Caesar’s Angel, the implication would be that the American girds on
his armor abroad but avoids conflict in his own back yard. Even so,
something of this impression may remain with the reader.

Responsibility at Home and Abroad

The pattern in which a nation allows its liberties to be subverted and
destroyed is a familiar one. It is often thought that certain peoples,
like the Germans and Russians, are more susceptible to
authoritarianism than others. One stereotype of the American—
descendant of Minute Men, the man who hates cops, the fan who
cherishes his right to boo the umpire—has contributed to the
impression that this submissiveness to authority has never been a
part of the American character. Lewis’s It Can’t Happen Here, Dos
Passos’s District of Columbia, and the novels about dictators in
southern states raise some doubts about the validity of this
fundamental assumption. Each of these novelists seems to feel that
the American citizen could well wake up one morning to find his
rights gone as did the German citizen of the early thirties. Lewis
dramatizes this catastrophe on a national scale, while Dos Passos,
Warren, and Langley present it on the state level. Dos Passos, all
through his trilogy, writes such frequent exhortations to vigilance that
there is little doubt that he too has worries on this score. In Stranger
Come Home Shirer centers his fire on McCarthyism, but he also
goes back into history to recall the passage of the Alien and Sedition
Acts and the domestic anti-German violence of World War I. The
popular press and magazines may represent the American as one
who hearkens to the great voices on each Fourth of July and
understands what he is doing when he raises his banner on Flag
Day. The novelist often has serious doubts that he does.
Europeans have called the United States immature in world affairs.
At least one of the novels in this group examines this accusation,
and two others consider the same theme of irresponsibility on the
national level. In both A Fool’s Errand and Bricks Without Straw
Tourgée accused the federal government of irresponsibility. There is
a close parallel between this case and the one Europeans have
made. Pursuing an ideal, at least in part, the United States musters
its enormous industrial and economic potential, puts its great
strength into the field, and wins military victory. After talking a great
deal about what should be done, it sets up committees which write
many reports. Then the nation promptly forgets the problems of
victory and happily returns to consideration of the tariff question or
who is going to win the World Series. In the latter of his two books,
Tourgée writes that the Northern statesmen and political writers
seemed always to assume that the destruction of slavery would cure
all the ills of the Negro. With a typical flourish, he adds:
The Nation gave the jewel of liberty into the hands of the [Negroes], armed
them with the weapons of self-government, and said: “Ye are many;
protect what ye have received.” Then it took away its hand, turned away
its eyes, closed its ears to every cry of protest or of agony, and said: “We
will not aid you nor protect you. Though you are ignorant, from you we will
demand works of wisdom. Though you are weak, great things shall be
required at your hands.” Like the ancient taskmaster, the Nation said:
“There shall no straw be given you, yet shall ye deliver the tale of bricks.”
Nearly seventy years later, in The Crack in the Column, Weller said
virtually the same thing. But this time both the problem and the
stakes were global.
The picture of American politics which emerges from the political
novel is an unflattering one. Although the “smoke-filled room” may be
a cliché in the newspapers, it is a fact in the novel. The deck of
candidates is shuffled, cut, and dealt—often from the bottom. The
national conventions are a combination of circus spectacle and
cynical chicanery. And after campaigns financed by funds from
special interest groups, the people’s chosen representatives get
down to the serious business of paying off their debts while lining
their pockets. The crusaders for liberty and justice who appear can
be set off against these political liabilities. But the electorate in
whose behalf they struggle often seems unaware of the importance
of the fight. Inheritors of a tradition of dissent and individual freedom,
they are fair game for demagogues. They are also easy marks for
the revolutionaries who, for perverted purposes, exploit their sincere
but naïve desire for social and economic reform. Although the
Republic somehow seems to weather periods of internecine violence
and reversion to authoritarian rule, its citizens have a bad case of
myopia in the field of foreign affairs. There are indications, though,
that the corrective lenses bought in two world wars are beginning to
bring distant events into focus. The American novelist’s view of his
own political arena agrees surprisingly well with many foreign
estimates of it. The American seems like an immature giant who
tolerates much rough behavior but rushes into conflict when he feels
that his basic security is threatened. Now the giant seems to be
settling down. Still likely to make violent moves, he is acquiring some
of the political sophistication that was, perhaps, too early expected of
him.

ITALY: A SELF-PORTRAIT
After a quarrel with Jean Colbert, Tony Maggiore in Caesar’s Angel
berates her friend Al Piazza: “You talk so big about it making no
difference between American and Italian girls. You ever hear a good
Italian girl open her mouth about politics? You hear her insulting the
men?” This assertion is contradicted by Stella in Silone’s A Handful
of Blackberries, but even were it completely true, it would be one of
the minor differences between American and Italian political behavior
patterns. Immensely different historical antecedents separate the two
peoples. The Italian, with a background of autocratic rule except for
comparatively short intervals, has played his political role on a far
different stage from that of the American. But there is more to it than
just politics. The economic bases which help to form political groups
have produced in Italy a stratification in which the layers are more
widely separated than any in America. The migrant fruit pickers, the
dust-blown Okies, the exploited miners in America seem well off
beside the systematically persecuted peasants and submerged city
lumpenproletariat of Italy. The Italians at the other end of the scale
are just as far from the norm. Gould, Fisk, and Vanderbilt may have
owned railroads, but Prince Torlonia owns immense ranges of the
Roman and Tuscan countryside, together with 35,000 acres of the
Fucino basin worked by eleven thousand farmers. But this great gulf
between classes is only one of the factors which appear responsible
for Italy’s political ills.
Silone concentrates mainly upon the peasantry in his novels. His
heart is close to them, and his description is sympathetic. But he
does more than set forth their sufferings. His books also diagnose
the cause of their problems and suggest solutions. Many of his
peasants appear like credulous, superstitious children. The four-day
thunder and lightning storm which nearly sweeps Pietrasecca off its
mountain in Bread and Wine is blamed upon two lovers who have
gone to live in a house considered damned. Don Paolo watches the
gathering of a crowd which is swept into such a hysteria by its roar of
“CHAY DOO! CHAY DOO! CHAY DOO!” that it forgets to listen to the
oracular radio voice it has come to hear. Silone pities them: “a
people whose wisdom was summed up in a few proverbs passed
down from generation to generation, had been literally submerged
and overwhelmed by propaganda.” Don Paolo also blames this
ignorance upon the Church in Italy. Like his teacher Don Benedetto,
who was said to have called the reigning pontiff “Pope Pontius XI,”
Don Paolo feels that Italy needs
A Christianity denuded of all mythology, of all theology, of all Church
control; a Christianity that neither abdicates in the face of Mammon, nor
proposes concordats with Pontius Pilate, nor offers easy careers to the
ambitious, but rather leads to prison....
Silone’s hero is equally dissatisfied with a religious vocation which
withdraws from life. He talks with the beautiful and spiritual Christina,
who devoutly waits to enter the convent:
Do you not think that this divorce between a spirituality which retires into
contemplation and a mass of people dominated by animal instincts is the
 source of all our ills? Do you not think that every living creature ought to
live and struggle among his fellow-creatures rather than shut himself up in
an ivory tower?
Although A Handful of Blackberries contains more scenes set in
Rome than the other two Silone novels discussed here, a better view
of the Italian city dweller is given by Taddei and Moravia. Taddei’s
The Pine Tree and the Mole is built about citizens of Livorno who are
as widely separated by wealth and position as are Silone’s Old
Zaccaria and the Tarocchi family. The circle of lawyers and politicians
who exchange wives and party labels are a different breed of men
from the jailbirds, pimps, and informers at the opposite extreme of
Livorno’s social structure. But both groups are equally adrift, both
caught in the same wave of post-World War I exhaustion and
economic derangement which sent so many Italians in search of the
answer that Mussolini seemed to provide. Taddei emphasizes
poverty and depression as the main factors which prepared the way
for the Fascist regime, but one senses something else, particularly
among his representatives of the educated class. They seem to be
seeking some sort of order, a stabilizing force in their political life.
Taddei says that the Italian Socialist Party seemed to offer the
solution. But the Socialists failed because
overwhelmed by the favorable-seeming course of events and overlooking
the most important phase of the matter, they lacked the time to think of
many things and, instead, viewed the period with optimistic eyes;
everything appeared to them to depend upon the number of adherents
that any particular event brought into their ranks. Political expediency thus
became, in a manner of speaking, epidemic, and the deepening crisis in
all its amplitude became apparent in the form of a spiritual crisis that was
its reflection.
Marcello Clerici in The Conformist finds his solution in Fascism.
Taddei’s hero turns at last to the same source of strength which
Silone singles out—the land and its people. They both feel, rather
like Orwell, that the masses are the ultimate source of their country’s
salvation. Old Lazzaro, shouting defiance at the Communists,
epitomizes these people: “There’ll always be someone that refuses
to sell his soul for a handful of beans and a piece of cheese.” In this
he is like Conrad’s Giorgio Viola, “the Garibaldino,” an old expatriate
whose divinities are Garibaldi and Liberty.
This small group of Italian novelists portray their countrymen as
members of widely separated economic and social classes. There
are the well-to-do who seem to seek order and get it with a
vengeance. And there are the poor of the cities and villages who are
the victims of this order, squeezed by poverty, by powerful
landowners, and harnessed by a church which renders more than is
his to Caesar, a church which brings politics into the confessional
and divorces religion from everyday life. Something of the spirit of
Garibaldi still lives, however, nourished by the deprived ones who
are close to the soil. Perhaps this is a modern political parable in
which the last shall be first.

SPAIN: AN AMERICAN PORTRAIT

Robert Jordan’s love of Spain is not an abstract emotion. He has a
deep feeling for its people, but he is still able to see their faults.
Throughout For Whom the Bell Tolls he makes conscious estimates
of their national character. He says they are a truly thoughtful and
considerate people who are not merely formally polite as are the
French. But they are also treacherous:
Of course they turned on you. They turned on you often but they always
turned on everyone. They turned on themselves, too. If you had three
together, two would unite against one, and then the two would start to
betray each other. Not always, but often enough for you to take enough
cases and start to draw it as a conclusion.
These characteristics cut across party lines. The Rebel slaughter
which left Jordan’s Maria orphaned and violated had an equally
brutal counterpart in the Republican massacre supervised by the
guerrilla chief Pablo in which twenty Fascists were beaten between
two lines of men and then flung over a cliff. Jordan thinks that killing
“is their extra sacrament.” Loving them, he tries to understand them:
There is no finer and no worse people in the world. No kinder people and
no crueler.... Forgiveness is a Christian idea and Spain has never been a
 Christian country. It has always had its own special idol worship within the
Church.... The people had grown away from the Church because the
Church was in the government and the government had always been
rotten. This was the only country that the reformation never reached. They
were paying for the Inquisition now, all right.
Occasionally one finds a Spaniard who seems almost a moderate,
like Frankie Perez in Adventures of a Young Man, but the most
persistent impression is one of a people who have a history of
misrule and violence, and who tragically turn to these very weapons
as the instruments of release from their consequences.

GREECE: AN AMERICAN PORTRAIT

George Weller’s Greeks in The Crack in the Column appear
surprisingly like Latins: “Each Greek is a volcano, and when he may
erupt no man knows, not even his friends who must quake with him,
not even himself.” In the number and complexity of their political
parties they resemble the French, yet in one way they seem to retain
something of the Greeks of antiquity viewing Imperial Rome. Small
and impoverished in the shadow of newly-risen giants, they cherish
their role as inheritors and transmitters of a great culture. At the
book’s end, Nitsa, like Molly Bloom in Joyce’s Ulysses, retires with
her reflections:
The whole world is philhellene, as proved by the retreating Germans
leaving a wreath on the Tomb of Ignotos before they roared north to die in
the ambushes of the Slavs. But most self-surrendering of the philhellenes
are the Americans. These young antiques deserve the most utter respect,
the kindest care.

FRANCE: A COMPOSITE PORTRAIT

The novels by Frenchmen in this study reveal relatively little about
national character and behavior. Stendhal deals with Italians and
Malraux primarily with Chinese. However, the ambitious Ferral, head
of the Franco-Asiatic Consortium in Man’s Fate may be revealing.
He hopes to make enough money in China to return home and buy
the leading French news-gathering and publicity syndicate. With this
power he hopes to regain office and “pit the combined forces of the
cabinet and a bought public opinion against the Parliament.” In
Malraux’s words one catches, under their cynicism, echoes of the
declining power reflecting upon vanished days of affluence:
The threat of bankruptcy brings to financial groups an intense national
consciousness. When their enterprises in distant corners of the world are
suddenly threatened with disaster they remember with mingled pride and
gratitude the heritage of civilization which their country has given them
and which they in turn have helped to pass on to colonial peoples.
In Presidential Agent Sinclair presents the French as a people who
have installed a rotten government to manage their affairs, a
government eager to join with Britain in obtaining temporary
surcease from German threats to its financial holdings by
hypocritically sacrificing Czechoslovakia. They had decided upon “a
compromise with Hitler as the cheapest form of insurance.” M. Denis
admits the resultant loss of power in Central Europe, but consoles
himself with the thought that “we still have North Africa and the
colonies, and we are safe behind our Maginot Line. Above all, we
don’t have to make any more concessions to revolution at home.”
In The Age of Longing Hydie Anderson reports Feyda Nikitin’s
deadly activities to Jules Commanche of the French Home Security
Department. A scholar and hero of the Resistance, he is one of a
new type, but there are not enough of them to fill “the sclerotic veins
of French bureaucracy with fresh blood.” Their effect amounts
merely to “the injection of a stimulant into a moribund body.” In The
Reprieve Sartre had presented France as unaware and unready
before the Nazis, already bled white from great wars. After another
conflict, the patient is almost in extremis. Playing the recurrent part
of American pupil to European teacher, Hydie listens to
Commanche’s bitter lecture:
Our last message to the world was those three words which are on our
stamps and coins. Since then, we no longer have anything to give to the
spirit, only to the senses—our novelists, our poets, our painters, all belong
to an essentially sensualist world, the world of Flaubert, and Baudelaire
and Manet, not to the world of Descartes, Rousseau and St. Just. For
 several centuries we were the inspiration of Europe; now we are in the
position of a blood donor dying of anemia. We can’t hope for a new
Jeanne d’Arc, not even for a young First Consul, not even for a Charlotte
Corday....
He has told Hydie that the French Revolution substituted its slogan
for the Holy Trinity, that the scalpel which excised autocracy from the
body politic also removed its soul. The French thus seem to be
suffering from a malady which, in somewhat different form, infects
the Spaniards, Italians, and Greeks. Their splinter parties, their
disorganization, their pervasive political cynicism are a legacy of
centuries of conflict, inequities, and colonial misrule. But beyond this
there is exhaustion, a vitality sapped by past efforts and a sadness
increased by awareness of faded glories.

RUSSIA: A COMPOSITE PORTRAIT

Assessing Russian national character or behavior from the novels
presents a greater problem than that found in any other national
literature. One must first separate Russian Communism from the
Russian people. In the same way one must distinguish the old
Russian from the new. And this dichotomy has to be made after the
Revolution as well as before it. Turgenev’s fathers are hardly more
different from their sons than are Koestler’s Old Guard from the
Neanderthalers they have sired. When Rubashov’s old comrade
Ivanov is shot, Gletkin replaces him as interrogator. Rubashov looks
at the shaven skull, appraising the huge ominous figure in the stiff
heavy uniform: “You consequential brute in the uniform we created—
barbarian of the new age which is now starting.”
The pattern of Russian political behavior which emerges from the
novel is filled with more violence, more misery, more oppression than
that of any other national group. The dying Stepan Verhovensky
described Russia in The Possessed as a “great invalid” inhabited by
devils and plagued with impurities, sores, and foul contagions. This
Czarist Russia is a land in which “harmless ... higher liberalism” is
possible, but it is also a country in which serfs live in incredible
poverty and aristocrats live in oriental splendor. Within this social
structure, whose opposite ends are separated by an even greater
distance than the rich and poor of Italy, the forces of destruction are
already at work. The basic situation reveals opposed characteristics:
authoritarianism on the ruling level, immense capacity for dumb
suffering in the submerged masses, and an intense drive toward a
reorientation of the social structure on the part of the militant
intellectuals. These factors, on a smaller scale, are to be found in
other literatures. But Dostoyevsky also portrays a conflict peculiar to
Russia. It is the struggle between those culturally oriented toward the
West and the Slavophiles who reject its influence. A Slavophile
himself, Dostoyevsky seems to speak through Shatov, who says that
the Russians are “the only ‘god-bearing’ people on earth, destined to
regenerate and save the world in the name of a new God, and to
whom are given the keys of life and of the new world....” This
extreme national consciousness pervades these novels, whether it is
expressed in terms of a messianic mission or a deep sympathy for a
people with a tragic history.
Conrad, in Under Western Eyes, made a definite effort to assess the
Russian character. He felt, though, that this was an extremely
difficult task in which the language barrier was the least of the
obstacles which stood in the way of understanding. He describes
Russians as great players with words, manipulators of abstract
ideas. He feels that in other areas their behavior, like that of Nathalia
Haldin, is sometimes almost incomprehensible. His narrator says:
I knew her well enough to have discovered her scorn for all the practical
forms of political liberty known to the Western world. I suppose one must
be a Russian to understand Russian simplicity, a terrible, corroding
simplicity in which mystic phrases clothe a naïve and hopeless cynicism. I
think sometimes that the psychological secret of the profound difference of
that people consists in this that they detest life, the irremediable life of the
earth as it is, whereas we Westerners cherish it with perhaps an equal
exaggeration of its sentimental value.
Nathalia tells the old teacher that “the shadow of autocracy” hangs
over each Russian, much as does its more substantial modern
counterpart, the Soviet MVD. Razumov is another Slavophile who
believes that Russia is sacred. But later Conrad returns to the
subject of Russian incomprehensibility when he says that Western
ears “are not attuned to certain tones of cynicism and cruelty of
moral negation, and even of moral distress already silenced at our
end of Europe.”
Koestler’s two novels considered here describe Russia after the
deluge. The bloodbaths have changed the names and faces, but the
same moral negation is there. More accurately, there is not even a
negation, for there is no positive assertion of moral values to negate.
The doctrine that history has no conscience had removed for the
Soviets the need for moral reference points. As a result, they had
sailed without the ethical ballast that Rubashov, at the end of his life,
decided was essential. In his judgment this was the fatal break in the
logical chain which caused the betrayal of the revolution which
Mailer mourned in Barbary Shore. In this case, perhaps, history is
character. The span of events covered by these novels might even
be graphed. The line would form itself into two low plateaus
separated by a single tremendous peak. This eminence would
represent the ill-fated revolt against tyranny. On either side would be
the depths in which a people was submerged with not quite passive
suffering under a rigid, repressive rule made possible by a long
historical pattern and the mass conditioning to obedience which it
produced.

UNION OF SOUTH AFRICA: A SELF-PORTRAIT

The Boers of South Africa are presented, especially in Paton’s
novels, as a simple and stern people who are also good fighters and
good haters. Subduing part of a continent and making it their own,
they regard themselves as the elect among the children of men quite
as much as do the Slavophiles. Yet these Afrikaners suffer from a
curious case of schizophrenia. Singled out by the Almighty, they are
nonetheless so fearful of the colored population that they repress
them as harshly as any European nation ever did its colonial
peoples. Early in Too Late the Phalarope Paton epitomizes this
national group:
 they had trekked from the British Government with its officials and its
missionaries and its laws that made a black man as good as his master,
and had trekked into a continent, dangerous and trackless, where wild
beasts and savage men, and grim waterless plains, had given way before
their fierce will to be separate and survive. Then out of the harsh world of
rock and stone they had come to the grass country, all green and smiling,
and had given to it the names of peace and thankfulness. They had built
their homes and their churches; and as God had chosen them for a
people, so did they choose him for their God, cherishing their
separateness that was now his Will. They set their conquered enemies
apart, ruling them with unsmiling justice, declaring “no equality in Church
or State,” and making the iron law that no white man might touch a black
woman, nor might any white woman be touched by a black man.
This is a patriarchal society, ruled by men such as Jakob Van
Vlaanderen, a political leader who privately calls the members of
Parliament “his span of oxen.” Fiercely nationalistic, they hate the
British, and as they fought them so they fight any force, even a
sociological one, which threatens their hard-won supremacy. In the
Union of South Africa there are willing British subjects and British
sympathizers, but they have not been represented by a group of
novels as fine as these of Paton’s. This is perhaps due to the fact
that they do not form so homogeneous a cultural and ethnic group as
do the Boers and their descendants. For another thing, the political
star of the Boers has been in the ascendant in the past few decades,
while that of the pro-British has seemed about to set as it did in
India.

GERMANY: A SELF-PORTRAIT
When Sinclair’s Lanny Budd goes to the week-long Nazi Party orgy
at Nüremberg, he sees before him a people who have surrendered
personal responsibility to a father image quite as fully as did Silone’s
simple peasants. A people smarting from humiliating defeat had
accepted the dream of a thousand year Reich. And it had been sold
to them by a man as possessed as any of Dostoyevsky’s characters.
Roderich Stamm in Heaven Pays No Dividends is oblivious to much
of the transition that takes place in Germany during the late twenties
and early thirties. Through his father’s conversations, however, he
senses some of the problems soon to be expressed in political
action: “They contained the whole uncertainty of our age. There was
something intangible and threatening in the air. It had all started with
the world crisis and the huge unemployment figures, and then the
Nazis had come, and then the Communists.” These forces and the
movements which they precipitate are too strong for a republic
barely fifteen years old. A people used to authoritarian rule reverts to
it. When war comes Roderich realizes “we were all involved,
because all of us had capitulated before HIM during all these years.
We had all given HIM permission to start a war at HIS discretion,
when HE decided it was necessary.” One is justified in distrusting
stereotypes as inaccurate generalizations. But in this case the
stereotype is borne out and emphasized in the novel.
Many areas of these national portraits are only roughed in, with the
fine-line detail missing. In other places there are gaps. This is partly
because some of the groups of novels are small. It is also due to the
fact that the novelist does not exhaustively examine voting trends,
statistically analyze attitudinal changes, or plot the frequency of
government realignments. Sinclair frequently approaches this
method and Dos Passos gives some of the raw data upon which
such estimates can be made. But generally the novelist tends to
proceed from individuals to groups, extrapolating group behavior
from individual behavior. This is the method used by Koestler and
Conrad. While it does not have the statistical validity of the political
scientist’s work, it has advantages which complement it. The
novelist, with his artistic insight and his ability to shape his material
as he wishes, can highlight his concept of a particular national
character with drama and human interest to make this hard-to-define
quality come memorably alive on the printed page.
 chapter five
The Novelist as Analyst of Group
Political Behavior
The economic criterion is one of two means used by the political
novel for classifying groups. The other index identifies groups by
means of overt political behavior—party membership, acceptance of
discipline, performance of specific acts. These two means of
classification are not parallel but complementary. The first, in a
sense, serves as a background for the second. Although the first is
economic, its validity lies in the fact that party lines tend to follow
economic ones, that modern political theory, particularly that of
revolution, has been based increasingly upon economic facts as well
as political ones. The approach of this chapter differs from that of the
previous one in tracing behavior patterns which cut across national
lines.

ECONOMIC GROUPS

The Lumpenproletariat
In trying to use these indices of group behavior it is no longer
possible to deal in general terms such as “the poor.” Though
“proletariat” may be precise enough for Marxism, even this term is
too inclusive for close analysis of the material in these novels.
Several gradations are possible within this least fortunate group on
the economic scale. Professor Ambrogio Donini’s introduction to the
Italian edition of The Pine Tree and the Mole identifies the lowest
group in that novel as members of the lumpenproletariat. These
people are not the workers whose taking of the factories Taddei
fleetingly mentions. They are the lowest stratum of Livorno’s life, the
drifters and criminals from whom the Fascists recruited members for
their Black Shirt squads. It is their motivation as much as their
behavior which differentiates them from the workers. While groups of
militant workers are usually presented as trying to better the lot of
their whole group, these members of the lumpenproletariat seem to
be entrepreneurs. Rubachiuchi becomes an agent provocateur for
the Fascists not to help the poverty-stricken, but simply to help
himself. After he infiltrates an anarchist group, he aids in its
destruction, not because he is personally opposed to anarchism, but
because this is the job his employers are paying him to do. Very
often in these novels one encounters Communists trying to destroy
Socialists ostensibly because Socialism is thought to be a palliative
rather than a solution to the worker’s problems. But these
underworld figures do not have even this theoretical justification.
Although this group is not nearly so numerous in the novel as many
others, its representatives occasionally appear. In Fontamara,
Peppino Goriano returns to Fontamara after thirty-five years, some
of them spent in a “political career” in Rome. A good man, he had
been forced by starvation to become an agent provocateur for the
police. He had earned five lire a day plus a twenty-five lire bonus
each time a job resulted in his going to the hospital. For a brief time
he had been a hero after a picture of him helping to wreck a
Communist newspaper office had appeared in a newspaper. But the
“Hero of Porta Pia” lost the honest job he had finally found when it
was decided that “fascism could no longer shelter in its bosom such
delinquents as had been convicted several times for theft.” Pablo in
For Whom the Bell Tolls is a study in deterioration. A cruel but
effective anti-Fascist at the beginning of the war, he is found by
Robert Jordan to be a sotted semi-bandit who opposes Jordan’s
blowing of the bridge. Having lost control of the band to his woman
Pilar, Pablo tries unsuccessfully to forestall the demolition by stealing
part of Jordan’s equipment. Although he leads the retreat after the
bridge is blown, Pablo is no longer one of the “illusioned ones.” He is
a guerrilla who retains only his violence and a dominating desire for
survival. Old Zaccaria in A Handful of Blackberries is a bandit first
and a partisan second. He had received a decoration for a crippling
encounter with a German patrol, but the source of the battle was a
truckload of cheese which he intended for the black market rather
than a desire to free Italy. These characters are not nearly such low
forms of life as some of the members of Taddei’s lumpenproletariat,
but they are a part of that group to be found on the fringes of most
political conflicts, individuals motivated by desire for personal gain
rather than by principle.

Peasants
Almost as submerged in the economic structure is the peasant class.
Here too there are extra-national similarities. The paisans of Silone
and Taddei and the muzhiks of Turgenev and Dostoyevsky have
characteristics in common. Ground down by oppression and
exploitation, they engage in political action only as a result of outside
stimulation rather than as a result of spontaneous desire among
themselves. Often they may be too devitalized even to do that. When
Bazarov in Fathers and Sons tells the peasants that they are the
hope of Russia, he does not suspect “that in their eyes he was all the
while something of the nature of a buffooning clown.” In a more
subtle way Don Paolo tries to awaken the peasants of Pietrasecca in
Bread and Wine. He has a little success with parables, but a direct
attack upon issues produces nothing. When he goes to Fossa he
asks the lawyer Zabaglione about peasant participation in the now
disbanded Socialist Leagues. Zabaglione tells him:
What Socialism meant to most of them was a chance to work and eat till
their stomachs were full, to work and sleep in peace, without having to be
afraid of the morrow. In the league premises at Fossa, next to the bearded
portrait of Karl Marx, there was a picture of Christ in a red shirt. On
Saturday nights the peasants came to the league to sing “Up, brothers!
Brothers, arise!” and on Sunday morning they went to Mass to say
 “Amen.” The permanent occupation of a Socialist leader was writing
recommendations.
The American representatives of this class seem much the same.
Garland’s A Spoil of Office follows the abortive political careers of
the Grange and the Farmer’s Alliance. Bradley Talcott sees this latter
movement as “the most pathetic, tragic, and desperate revolt against
oppression and wrong ever made by the American farmer.” His
guiding star Ida puts it even more simply: “While our great politicians
split hairs on the tariff, people starve. The time has come for
rebellion.” Conditions in Iowa have changed eighty years later, but in
the deep South they are almost as bad. William Russell’s A Wind Is
Rising (1950) focuses on Negro tenant farmers charged 500 per cent
interest by the landowners and cheated of part of the cotton crop
they succeed in raising.
Whether he is Prince Torlonia or a Russian noble, the large
landholder is most often seen in the novel as the embodiment of the
forces which keep the peasant class in subjection. Men like Nikolai
Kirsanov in Fathers and Sons may attempt to improve conditions,
but the predominant pattern is one of exploitation which eventually
produces violence. At the end of A Handful of Blackberries the
peasants kill a bailiff when they invade the Tarocchi pasture lands
they believe to be rightfully theirs. Debased and deprived, lacking
political awareness, and incited by members of other classes, the
peasant group turns to violence. In some areas of the world,
particularly the United States and England, a steady economic
evolution has tended to eliminate large segments of this group. The
Russian peasant appears still to be a peasant, even though he may
be a Stakhanovite worker in a large kolkhoz. But where the class still
exists in a society in which violent protest is still possible, the pattern
appears unchanged.

Labor
Commenting on one of the causes of the failure of the farmers’
movements of the seventies, Garland said: “They had made the