Review TRENDS in Microbiology Vol.12 No.
6 June 2004
Error-prone replication for better or
worse
Brigette Tippin, Phuong Pham and Myron F. Goodman
Hedco Molecular Biology Laboratories, Departments of Biological Sciences and Chemistry, University of Southern California,
Los Angeles, CA 90089-1340, USA
Precise genome duplication requires accurate copying
by DNA polymerases and the elimination of occasional
mistakes by proofreading exonucleases and mismatch
repair enzymes. The commonly held belief that ‘if some-
thing is worth doing, then it’s worth doing well’ nor-
mally applies to DNA replication and repair, however,
there are exceptions. This review describes elements
that are crucial to cell fitness, evolution and survival in
the recently discovered error-prone DNA polymerases.
Large numbers of errant DNA polymerases, spanning
microorganisms to humans, are used to rescue stalled
replication forks by copying damaged DNA and even
undamaged DNA to generate ‘purposeful’ mutations
that generate genetic diversity in times of stress. Here
we focus on low-fidelity polymerases from bacteria,
comparing Escherichia coli, archeabacteria and those
most recently discovered in Gram-positive Bacilli,
Streptococcus, pathogenic Mycobacterium and intein-
containing cyanobacteria.
Negative consequences of mutations, including human
hereditary and sporadic diseases, are counterbalanced
by the requirement for mutations in evolution, fitness
and immunological diversity. Except for the immune
response, mutations influence microorganisms and
higher organisms comparably – almost all are non-
beneficial. Mutations are an apt reflection of Damon
Runyon’s* succinct observation that ‘all life is six to
five against’ [1].
Simple mutations, including base substitutions and
small frameshifts, are categorized as either spontaneous
or induced. High-fidelity (HiFi) replicative DNA poly-
merases, which are far from perfect, generate spontaneous
errors when copying DNA, with mutation rates ranging
from 1024 to 1025 per base pair. Many replicative
polymerases have an associated proofreading exonuclease
that excises perhaps 90 to 99 out of a total of 100
misincorporated nucleotides, reducing spontaneous poly-
merase error rates to within the range of 1025 – 1027.
Corresponding author: Myron F. Goodman (mgoodman@usc.edu).
* Damon Runyon, a renowned short-story writer and New York sports columnist in
the 1920s–30s and whose Broadway stories inspired the hit show and motion picture
‘Guys and Dolls’, died as a result of throat cancer in 1946. His ashes were scattered out
of a plane over Broadway by World War I air ace Eddie Rickenbacker. Aside from the
‘all life is six to five against’ quotation, Runyon continues to play a central, albeit
ironic, role in the relation of mutation with human disease owing to the Damon
Runyon Cancer Research Foundation that identifies and supports young US scientists
committed to discovering the causes and cures for cancer.
www.sciencedirect.com 0966-842X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.04.004
Replication errors can also originate as a result of different
types of damage, for example, the spontaneous deamina-
tion of cytosine to uracil or by exposure to oxygen,
radiation or chemicals. Standard issue high-fidelity DNA
polymerases are often hindered when confronted by
damaged template bases, causing replication to stall.
The resulting inability to copy the entire genome inevi-
tably causes cell death. However, this deficiency can be
alleviated by calling on low-fidelity error-prone (EP)
polymerases to do the translesion synthesis (TLS) ‘dirty
work’ because EP polymerases are able to copy damaged
DNA templates permissively and efficiently [2].
There are surprisingly large numbers of these errant
copiers, two of which were first recognized in yeast (Rev 1)
and Escherichia coli (polymerase V), followed shortly by
numerous homologs in microorganisms and approxi-
mately a dozen homologs in humans [2]. The use of EP
polymerases can be a double-edged sword. Although
replication past damaged DNA templates can help to
alleviate cell death, the cost associated with a concomitant
increase in mutations can be considerable. Typical EP
polymerase error rates on undamaged DNA templates
range from 1021 to 1023 per base pair; several orders of
magnitude greater than the HiFi replicative polymerases.
It is therefore necessary for cells to regulate both where
and when EP polymerases come into play to reduce the
chance that mutations occur when copying undamaged
DNA. In Gram-negative E. coli, there are three damage-
induced polymerases, and although each plays a role in the
well-being of the cell, all are dispensable [3]. By contrast,
EP polymerases in some Gram-positive bacteria are
essential for survival, perhaps as an integral part of the
replication complex [4,5].
Two general biological themes have been developed to
reflect two possible pathways for endogenous mutagen-
esis. The first relates to cellular regulation of mutagenesis,
which is exemplified by the well-established SOS pathway
in E. coli. In this instance, three dispensable EP
polymerases enhance survival following the induction of
DNA damage and also help to ensure competitive fitness
by generating genetic diversity. A second theme relates to
the presence of indispensable EP polymerases in Gram-
positive cells as an integral part of the replication complex.
This review discusses a variety of EP bacterial poly-
merases that are used to perform efficient TLS and reflects
upon the biological consequences, either for better or
worse.
 Review TRENDS in Microbiology
SOS-induced E. coli DNA polymerases
The SOS response (Box 1) is responsible for inducing more
than 40 genes, enabling E. coli to cope with UV or
chemically induced chromosomal damage. A considerable
number of SOS-induced enzymes are engaged in removing
aberrant DNA structures by nucleotide excision and base
excision repair, whereas replication and recombination
enzymes are used to rescue stalled replication forks [6].
These 3R (repair, replication and recombination) path-
ways exhibit essentially no increase in mutations above
spontaneous background levels. In contrast to error-free
3R pathways, SOS polymerases are principally involved in
error-prone TLS pathways, typically causing large
increases in mutations. Therefore, the SOS response is
divided into two pathways to process DNA lesions: first,
high-fidelity (error free, 3R repair, primarily using non-
inducible polymerases I and III) and the other low-fidelity
(error-prone, using the three SOS-regulated polymerases
II, IV and V). However, there is not a strict division of labor
for each pathway. Polymerase III, for example, is needed to
replace an EP polymerase and complete genome replica-
tion after a damaged DNA nucleotide has been copied,
whereas polymerase II performs TLS in an error-prone
manner but also plays a role during error-free replication
restart.
Box 1. Regulation of error-prone polymerases during the SOS stress response in Escherichia coli
Figure I depicts the regulation of the cellular response to DNA damage in
Escherichia coli, termed the SOS response [62]. More than 40 genes are
upregulated upon exposure to DNA-damaging agents, such as
chemicals and UV light [63]. The regulator for SOS induction is a two-
component repressor and activator system of LexA and RecA proteins.
The LexA repressor binds to a 20 base pair consensus sequence in the
operator region of the SOS genes, suppressing their expression. The
SOS response begins once RecA becomes activated by forming a
nucleoprotein filament on single-stranded regions of DNA, perhaps
created by blockage of replication forks. The activated RecA* nucleo-
protein filament serves as a co-protease that facilitates the autocatalytic
cleavage of the LexA repressor. Cleavage of LexA in turn activates
transcription of all lexA-controlled genes. Early upregulated genes
following SOS induction include genes involved in nucleotide excision
repair uvrA, uvrB and urvD and genes involved in recombination repair
recA, recN, ruvA and ruvB [20]. One of the earliest induced genes (, 1
LexA
recA polB
<1 min <1 min
RecA
DNA damage pol II
LexA RecA*
5′
Figure I. The regulation of cellular responses to DNA damage in Escherichia coli.
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Vol.12 No.6 June 2004 289
E. coli has developed an effective strategy to regulate
EP polymerase expression using the SOS regulon, with
DNA polymerases II, IV and V induced in response to DNA
damage (Box 1). Polymerases IV and V, members of the
recently discovered Y-family [7], exhibit broad-specificity
TLS and copy undamaged DNA with extremely poor
fidelity [8]. Polymerase II, a third SOS-induced polymer-
ase, can perform error-prone TLS in vivo [9], but executes
high-fidelity synthesis on undamaged templates, akin to
replicative polymerase III. Base substitutions and small
deletions are the most abundant types of SOS-induced
mutations. Base substitutions are generated by a two-step
mechanism in which a nucleotide is misincorporated
directly opposite a damaged DNA nucleotide followed by
accurate DNA synthesis downstream from the lesion
(Box 2); this is the favored mode of action for polymerase
V TLS in vivo (Table 1). Alternatively, small deletions can
occur by skipping the lesion on a transiently misaligned
primer-template strand (Box 2). This latter mechanism
can be used by polymerase IV when making 2 1 deletions
[10], whereas other DNA slippage mechanisms can cause
larger deletions [11]. In vivo, polymerase IV generates
small non-targeted frameshift mutations [12,13], which
might occur when helping to rescue stalled replication
forks on undamaged DNA [6], and is also involved in
minute after SOS induction) is polB encoding E. coli DNA polymerase II.
Among the latest expressed genes are umuD and umuC (, 45 minutes)
and sulA, encoding a cell division inhibitor. Functions of more than 20
other lexA-dependent SOS genes remain to be characterized.
One consequence of SOS induction is an approximate 100-fold
increase in mutations above spontaneous levels. SOS-induced
mutations occur primarily at sites of DNA damage. This phenomenon
is known as SOS mutagenesis or SOS error-prone repair [62]. Genetic
studies have shown that SOS mutagenesis is dependent on the
expression of two genes, umuD and umuC (named after uv-induced
mutagenesis). In addition, UmuD protein must undergo RecA*-
mediated autocleavage (similar to LexA autocleavage) to produce a
shorter, mutagenetically active UmuD0 . Two UmuD0 molecules combine
with one UmuC molecule to form a heterotrimeric UmuD20 C complex,
identified as error-prone DNA polymerase V [16 –18]. Another poly-
merase induced in response to DNA damage is polymerase IV (dinB).
dinB umuDC
? ~45 min
pol IV
UmuC
UmuD
UmuC
D′ D′
UmuD′ pol V
TRENDS in Microbiology
290 Review TRENDS in Microbiology Vol.12 No.6 June 2004

distinct from its role as a co-protease in the cleavage of

Box 2. Translesion synthesis (TLS) is highly error-prone LexA and UmuD proteins [21,22] (Box 1).
DNA synthesis on an undamaged DNA template normally proceeds The ability of RecA to assemble cooperatively and form a
with high efficiency and high fidelity by DNA polymerases. However, nucleoprotein filament on exposed single-stranded (ss)
when a lesion (X) is present, replication efficiency and fidelity are DNA regions [20], which might form as a result of
reduced severely because of the miscoding nature of most lesions.
continued unwinding of DNA by the DnaB helicase
DNA polymerases copy damaged DNA templates (termed transle-
sion synthesis or TLS) by inserting one of the four dNTP substrates downstream of a stalled replication fork (Figure 1), could
across the damaged base followed by extension beyond the damage provide a common structural feature connecting recombi-
site. The process of TLS is markedly error-prone (see Table 1 in the nation, SOS induction and mutagenesis. However, DNA
main text). As most lesions completely alter the pairing properties of polymerases, including polymerase V, cannot copy tem-
the pre-existing bases, the polymerases probably insert incorrect
nucleotides opposite the lesions, therefore giving rise to base plate strands on which intact RecA filaments have formed
substitution mutations, for example, polymerase V (Figure I). [19]. The inhibitory filament effect is relieved by the action
Some DNA polymerases, for example, Escherichia coli polymerase of a single-stranded binding protein (SSB) in conjunction
IV, prefer to skip past lesions, inserting a correct nucleotide opposite with polymerase V, which strips RecA off the DNA in a
bases downstream from the lesion, generating frameshift mutations
(Figure I).
manner loosely analogous to a locomotive cowcatcher† [19]
(Figure 1). However, polymerase V-catalyzed TLS can also
occur in vitro on short ssDNA templates that are too small
T•A•G•C•
to support RecA filament formation [23]. Although RecA
A•T•C•G•X•T
must be present for polymerase V to copy damaged DNA,
assembly of a RecA filament per se is not required for TLS
pol V pol IV
in vitro [23]. The biochemical data introduce a ‘fly in the
T•A•G•C•A T•A•G•C• A
A•T•C•G T•C
filament ointment’, suggesting that there could be two
A•T•C•G•X•T • •
X distinct modes of RecA action: one to activate polymerase V
Base substitution
or Error-free Frameshift
directly [23] and a second to bind to DNA that is proximal
to a lesion and shepherd polymerase V past the damaged
TRENDS in Microbiology site [24,25]; neither mode demands assembly of a RecA
nucleoprotein filament (Figure 2).
Figure I. Translesion synthesis.
From a mechanistic perspective, it appears that RecA
mode 1 is essential for TLS, but the requirement for a
copying bulky template adducts (Table 1) [9,14,15]. Both
distinct second RecA mode remains open. A point to keep
polymerase II and IV play a central role in generating
in mind, however, is that although the biochemical data
adaptive mutations in non-proliferating microbial popu-
can be used to define the simplest system that might reflect
lations that face non-lethal selective pressure (Table 1) [2].
many salient features of TLS, the actual biological process
is typically more complex. For this reason, it cannot be
Biochemical basis of translesion synthesis in E. coli
ruled out that a RecA filament participates in TLS in vivo,
Actions and interactions of polymerase V and RecA
possibly by targeting polymerase V to the lesion site,
The induction of chromosomal mutations during the SOS
displacing polymerase III core from the b processivity
response requires the combination of UmuC, containing
clamp (Figure 1), and by stabilizing a RecA moiety at the
the catalytic domain of polymerase V [16,17], and cleavage
30 -filament tip. In vivo requirements include the b clamp,
of UmuD to UmuD0 (Box 1) to form the mutagenically
which serves as a common polymerase docking point and
proficient polymerase V complex, designated UmuD20 C
central element in polymerase swapping, and SSB to help
[18]. However, polymerase V is essentially dead in the
disassemble the filament ahead of the advancing poly-
absence of RecA protein, which stimulates its activity more
merase V.
than 100-fold in vitro [19]. RecA is a multitask protein that
is central to homologous recombination [20], the induction
Trading places
of SOS (Box 1) and also SOS mutagenesis caused by TLS.
The presence of five unique polymerases in E. coli, each
The latter mutagenic property of RecA is genetically
with their own specificity, sparks the question of traffick-
Table 1. Mutations generated during translesion synthesis ing the right polymerase at the right time to a DNA
(TLS) in Escherichia coli are dependent on the nature of lesions molecule. E. coli b clamp binds all five DNA polymerases
and polymerases and also the MutS mismatch repair protein and ligase [26],
whereas the eukaryotic proliferating cell nuclear antigen
Lesiona TLS polymerase Mutation
(PCNA) has an even wider range of binding partners,
Abasic site [8,10] pol V A, Gb
pol IV 2 1 frameshift including the replication, repair and cell-cycle checkpoint
6-4 TT Photoproduct [8] pol V T!C proteins [27]. The site on b clamp that is required for
BaP [9,14] pol V and pol IV 2 1 frameshift interaction with the replicative polymerase III a subunit
AAF [9] pol V 2 1, 2 2 frameshift [28] was recently shown to be the same site that governs
pol IV 2 2 frameshift
pol II 2 2 frameshift
† A cowcatcher, commonly used in the midwestern United States, is a triangular
8-oxo-dG [15] pol V and pol IV 2 2 frameshift
metal grill at the front of a locomotive that pushes meandering stray cows off a
a
BaP, Benzo(a)pyrene; AAF, N-2-acetylaminofluorene; 8-oxo-dG, 8-oxo-guanine. railroad track: polymerase V, SSB and RecA represent the locomotive, cowcatcher and
b
Preferential incorporation of dAMP and dGMP opposite the abasic moieties. cows, respectively.

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 Review TRENDS in Microbiology Vol.12 No.6 June 2004 291

SSB
pol III
core
β

Assembly of pol V mutasome complex

polymerase switching
DnaB

pol III
core

SSB RecA∗
RecA
pol III pol III
Lagging core core pol V
strand τ τ
β-clamp β
β
RecA

Disassembly of pol V mutasome complex

after TLS, pol III resumes replication

γ-complex
pol V
Leading
strand
pol III
core
β

TRENDS in Microbiology

Figure 1. Polymerase swapping alleviates the blockage of replication forks by DNA template damage. Forward motion of a replication fork (shown at the left), is blocked
when the polymerase (pol) III replication complex encounters a damaged DNA base (jagged line) on the leading template strand. Unwinding of DNA ahead of the lesion by
DnaB helicase could result in a stretch of single-stranded DNA that can support assembly of a RecA nucleoprotein filament, further inhibiting movement of polymerase III.
The replication complex composed of polymerase III core, b processivity clamp, g clamp-loading complex and t dimer, dissociates and the polymerase III core, which was
originally tethered to b, is replaced by error-prone polymerase V (shown at the right). A RecA moiety bound to polymerase V (‘mutasome’ complex) is required for transle-
sion synthesis (TLS). The presence of a RecA nucleoprotein filament (RecA*) inhibits TLS unless single-stranded binding protein (SSB) is also present to enable stripping of
RecA from the template strand, in a 30 to 50 direction. Once TLS has occurred, polymerase V is replaced by the polymerase III core, the replication fork is re-started and syn-
thesis is resumed on undamaged DNA downstream from the lesion.

interaction with polymerase IV [29,30] and the UmuC
subunit of polymerase V [28], arguing that only one
partner can interact with b clamp at once, depending on
the replication context. Regulating the level of polymerase
V is especially crucial as it is extremely noxious to the cell.
Its access to DNA is modulated by the combination of late
SOS induction, post-translational processing of UmuD by
Lon and ClpXP proteases [31,32] and subsequent
exchange of UmuD for cleaved UmuD0 [33] to make a
functional polymerase V complex (UmuD20 C; Box 1). All of
these events occur as an automatic consequence of the
triggering of the SOS pathways.
post-SOS induction, are below detectable levels (,15 per
cell) in uninduced cells [36]. What roles might polymerases
II and IV have and how do their normal levels influence
their ability to compete for b clamp? Polymerase IV is
responsible for most adaptive mutations, typically 21
deletions, in slowly dividing or non-dividing stationary
phase cells placed under non-lethal selective pressure
[37,38], whereas polymerase II, through its 30 -exonuclease
RecA, mode 2 (?)
pol V

Life in the slow lane: a role for SOS polymerases in fitness

and evolution
Although the SOS polymerases are induced in response to
DNA damage, two of the three polymerases are also
present at significant constitutive levels. Polymerase II is
maintained at , 50 molecules per cell, increasing by
sevenfold following SOS induction [34,35], whereas con-
stitutive polymerase IV levels consist of , 250 molecules
per cell, increasing by tenfold when SOS is turned on [13].
By contrast, polymerase V levels, present at , 200 per cell
RecA, mode 1
TRENDS in Microbiology
Figure 2. Model depicting two modes of RecA action required for polymerase (pol)
V-catalyzed translesion synthesis (TLS). In mode 1, RecA binds to polymerase V at
a 30 -primer end resulting in a greater than 100-fold stimulation of polymerase V
activity. In mode 2, a RecA moiety is bound to the template strand at the 50 side of
the lesion to facilitate TLS. RecA mode 1 is necessary, but perhaps not sufficient,
for polymerase V-catalyzed TLS on short single-stranded DNA regions that cannot
support RecA filament assembly. The possible involvement of a second RecA
mode remains open.

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292 Review TRENDS in Microbiology
Table 2. Representatives of error-prone DNA polymerases in Prokarya and Archaea
Polymerasea Organism
Family B
Pol II (AAC73171) Escherichia coli
Family C
DnaE (NP 607343) Streptococcus pyogenes
DnaE2 (NP 217887) Mycobacterium tuberculosis
Family Y
Pol IV (Q47155) Escherichia coli
Pol V (AAA98073 and AAA98074) Escherichia coli
Dbh (T46875) Sulfolobus solfataricus
Dpo4 (NP 343798) Sulfolobus solfataricus
a
Genbank accession numbers are shown in parentheses.
b
Error rate per base pair.
proofreading function, helps reduce the magnitude of
adaptive mutations by approximately fivefold [39]. The
contributions of polymerases II and IV to adaptive
mutation in vivo exemplify a more general phenomenon
in which each SOS polymerase can establish a transient
mutator phenotype, helping cells to cope with adverse
environmental conditions [40]. Cells lacking any SOS
polymerase that are grown for long periods in competition
with wild-type E. coli exhibit a severe reduction in long-
term survival and evolutionary fitness [3]. Mutations
could also arise by error-prone copying of so-called cryptic
DNA lesions that arise from endogenous spontaneous
DNA damaging events.
Gram-positive bacterial EP polymerases
Family C DnaE polymerases
Family C polymerases are homologs of the E. coli poly-
merase III a catalytic subunit (encoded by the dnaE gene),
which is responsible for chromosomal replication in Gram-
negative organisms [41]. In Gram-positive bacteria, two
distinct subclasses of family C have been identified: class I
(dnaE-type), which lack proofreading abilities; and class II
( polC-type), which also encode an intrinsic 30 – 50 proof-
reading exonuclease. In Bacillus subtilis and Staphylo-
coccus aureus, PolC is believed to work on the leading
strand and DnaE works on the lagging strand, whereas in
other bacteria DnaE is responsible for leading and lagging
synthesis [4,5].
Streptococcus pyogenes contain class I (DnaE) and II
(PolC) family C polymerases, which are both essential for
cell viability and use the b-sliding clamp [42], but only
PolC has been established as the replicative enzyme.
S. pyogenes DnaE, however, has been recently character-
ized as highly error-prone, producing frameshifts and
point mutations on undamaged DNA and during TLS
in vitro (Table 2) [43]. In contrast to the previously
characterized family C polymerases, S. pyogenes DnaE
appears to play a role that is similar to that of family Y
polymerases in E. coli, bypassing damaged DNA to
promote cell survival, except that family Y polymerases
are dispensable [3].
The pathogen Mycobacterium tuberculosis has two class
I polymerases, known as DnaE1 and DnaE2, but lacks
PolC [5]. Deletion of dnaE1 is lethal and is therefore
presumed to be the replicative polymerase, whereas
dnaE2 mutations do not affect survival [44]. In a screen
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Vol.12 No.6 June 2004
Error rateb Functions
1025 –1026 TLS, replication restart, adaptive mutation
1022 –1023 Chromosome replication, TLS
? Chromosome replication, TLS
1023 –1024 TLS, adaptive mutation
1022 –1023 SOS mutagenesis, TLS
1022 –1023 ?
1022 –1024 ?
for UV-damage-induced genes [44], dnaE2 was elevated
through an SOS-type response under lexA regulation,
whereas family Y genes dinP and dinX (both polymerase
IV homologs) failed to respond [44,45]. Therefore, a unique
SOS response has evolved in Gram-negative versus Gram-
positive bacteria, with LexA regulating distinct polymer-
ase families in each. The UV-induced mutations in vivo in
M. tuberculosis were characteristic of error-prone TLS
across cyclobutane pyrimidine dimers or pyrimidine-
pyrimidone photoproducts and were strictly dependent
on the presence of DnaE2 (Table 2) [44], as is the case for
polymerase V in E. coli. Overexpression of DnaE2 in the
absence of exogenous DNA damage fails to produce
mutations above background frequency, arguing it cannot
act alone to access undamaged DNA [44]. However, DnaE2
lacks a consensus b clamp-binding motif [46] and therefore
might rely on other damage-response proteins that are
also upregulated during the stress-induced response.
Similar to polymerase V during the SOS response,
M. tuberculosis DnaE2 and RecA exhibit temporal SOS
regulation, with peak induction around 60 minutes post-
irradiation.
Notably, the ability of M. tuberculosis to persist and
develop antibiotic resistance during infection is achieved,
at least in part, through this dnaE2- dependent muta-
genesis pathway [44]. Following infection, an immune
response containing reactive oxygen and nitrogen inter-
mediates might trigger continual SOS induction in
M. tuberculosis. Mice infected with a dnaE2-knockout
strain had tenfold lower colony counts from lungs than the
wild-type parental strain and exhibited 57% longer
median survival times (384 versus 222 days) post-infec-
tion. The number of rifampicin-resistant mutants that
emerged from mice infected with wild-type strains
included up to 18 isolates, whereas only one drug-resistant
mutant was recovered from the dnaE2-knockout-infected
mice [44].
Multiple copies of dnaE genes have been found in other
Gram-positive bacteria, including other Mycobacteria and
Streptomyces, and also in Gram-negative bacteria, includ-
ing Xanthomonas, Agrobacterium tumefaciens, Sinorhizo-
bium meliloti, Mesorhizobium loti and Pseudomonas
aeruginosa. All of these genomes also contain one or
more dinP –dinB homolog [44], which is analogous to
E. coli polymerase IV [7]. It remains to be determined
whether these organisms use their second dnaE gene
 Review TRENDS in Microbiology
for TLS mutagenesis and survival under stress rather
than dinP.
Since the discovery that dnaE2 is part of the SOS-
induced regulon in M. tuberculosis, the question of the role
of dnaE in B. subtilis has been revisited. Le Chatelier et al.
[47], unable to delete dnaE because of lethality, instead
reduced the effective concentration of DnaE in B. subtilis
and found a marked reduction in UV-induced mutagen-
esis. An SOS box is located 74 base pairs upstream of the
dnaE gene, the activity of which is induced threefold
following DNA damage and exhibits dependence on DinR,
the SOS repressor in B. subtilis [47]. In B. subtilis, it is
possible that DnaE plays a role in replication and TLS
mutagenesis, depending on protein interactions in the cell,
but how this is coordinated remains unclear.
In addition to novel TLS capabilities, another feature of
interest surrounding DnaE proteins is found in the
cyanobacteria Trichodesmium erythraeum (Genbank
accession numbers ZP_00075043, ZP_00074992 and
ZP_00074086) and Synechocystis (Genbank accession
numbers NP_440562.1 and NP_443058.1); specifically
the presence of inteins embedded in their coding sequences
[48,49]. Inteins are co-translated along with dnaE, but
once translated the inteins become enzymatically active
and catalyze protein-splicing reactions that result in their
own removal from the protein. The inteins then join the
flanking peptide sequences to produce functionally active
DnaE. Inteins have been found in many different proteins,
ranging from bacteria to archea and eukaryotes, demon-
strating an evolutionary origin as mobile genetic elements.
It remains unknown if the intein-containing DnaE
polymerases exhibit TLS activity, but if so, it could add
another level of complexity to the regulation of mutagen-
esis within the cell.
Family Y polymerases
The Y family EP polymerases are found in a wide variety of
organisms, from Gram-negative and Gram-positive bac-
teria to archeabacteria and eukaryotes [7]. In E. coli, the Y
polymerases are encoded on the chromosome, whereas in
other organisms they are associated with a variety of
mobile genetic elements, including plasmids and transpo-
sons [50]. No homologs of E. coli UmuD have been found in
Gram-positive bacteria, but it remains to be determined if
a functional substitute exists or is required. A wide variety
of harmless and pathogenic Gram-positive organisms,
including B. subtilis, M. tuberculosis, Bacillus anthracis,
Enterococcus feacalis, Lactococcus lactis, Corynebacter-
ium diphtheria and Streptococcus pneumoniae, contain
family Y homologs, but whether these putative EP
polymerases are functional or play a role in the evasion
of host immune systems is not known [50].
Error-prone family Y polymerases in Archaea
Sulfolobus solfataricus archeabacteria contain family Y
homologs that are most closely related to E. coli DNA
polymerase IV (dinB) [7]. The S. solfataricus strain P1
encodes a Dbh polymerase (dinB homolog) and a second
homolog Dpo4 has been found in the closely related but
distinct P2 strain of S. solfataricus [51]. Both Dbh and
Dpo4 exhibit low fidelity, can perform TLS across a variety
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Vol.12 No.6 June 2004 293
of lesions and cause frequent frameshifts in undamaged
and lesion-containing DNA in vitro (Table 2) [51– 53]. Dbh
and Dpo4 were the first family Y polymerases to be
crystallized [52,54,55], providing insight into the mech-
anism of low-fidelity synthesis in family Y. The structures
revealed a large active site that makes limited contact with
the DNA but also provides room for catalysis in the context
of bulky lesions, mismatches and in the case of the dinB
homologs extension from misaligned primer termini. The
bi-substrate crystal structure also showed that an incom-
ing nucleotide could pair with either of two template bases
in the active site, allowing for slippage to occur [54].
S. solfataricus Dbh can use PCNA and replication
factor C (RFC) during replication [56], whereas poly-
merase IV from E. coli uses the b clamp and clamp loader
accessory proteins during synthesis. The replication
machinery in Archaea is more closely related to Eukarya
than Prokarya, therefore the family Y polymerases have
evolved to interact with the available replication
accessory proteins in their respective environments. A
Dbh mutant in the phenylalanine residue at position 12
eliminates discrimination against rNTPs in vitro, which
become incorporated as readily as dNTPs, even during
frameshifts [57]. Dbh and Dpo4 can also incorporate and
extend from oxidized nucleotides, exclusively incorpor-
ating 8-oxo-dG opposite template A and preferentially
incorporating 2-oxo-dA opposite template G over tem-
plate T [58]. Family Y polymerases might prove to be
biologically mutagenic not only through TLS, but also
through incorporation of bases from adulterated pools of
nucleotides in the cell.
Perspectives
EP polymerases rescue stalled replication forks by copying
damaged DNA bases that block normal DNA synthesis,
leaving a sizable mutational load in their wake. Mutations
targeted to lesions might simply represent the cost of
completing the replication of the genome: mutate and
survive versus incomplete replication and death. EP
polymerases also occasionally copy either undamaged
DNA or DNA containing lesions that have been generated
endogenously. These untargeted mutations in bacteria
offer significant flexibility, enabling a cell to cope more
effectively in times of stress and to evolve.
Homologs of the EP polymerases described here exist in
several eukaryotic organisms, ranging from yeast to
humans, and have recently been reviewed in Ref. [2].
Notably, in eukaryotes not all EP polymerases cause
mutations as a result of TLS. For example, the human
family Y homolog DNA polymerase h performs TLS past a
T-T cyclobutane pyrimidine dimer by inserting two A
residues, resulting in the lesion being bypassed in an
error-free manner and still exhibiting overall low fidelity
on undamaged DNA [59]. The presence of polymerase h
protects cells from UV damage and a form of Xeroderma
Pigmentosum, in which patients have a higher risk to skin
cancer in the absence of the polymerase [60]. By contrast,
DNA polymerase i, another human family Y member,
achieves TLS by a highly error-prone bypass of a T-T
cyclobutane pyrimidine dimer [61]. As in bacteria, the
eukaryotes must also regulate access of EP polymerases to
294 Review TRENDS in Microbiology
DNA, ensuring the appropriate polymerase is called upon
for TLS at the right time.
The replication, repair and recombination pathways
used to regulate and maintain genomic integrity appear
strongly coupled, and a major challenge is to determine
how, when and where each enzyme is used. A reasonable
starting point is the sliding processivity clamps that help
regulate enzyme trafficking. When targeted to lesions, are
the various EP polymerases afforded unfettered random
access to the clamp proteins, which act to stabilize a
particular EP polymerase depending on the nature of the
impediment? Answers to this and closely related questions
of choice, such as how replicative and EP polymerases are
swapped to and fro, are tractable experimentally using
model biochemical systems. Although the devil, as always,
is in the details, this tried and true biochemical approach
to reconstitute competitive TLS at a replication fork in a
test tube should help furnish initial insights into the
factors governing the when, where and, perhaps most
importantly, how of polymerase switching.
Acknowledgements
We thank the NIH GM 21422 and ES012259 for research support.
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