Replication of DNA and

Chromosomes
 Chapter Outline
Basic Features of DNA Replication In
Vivo
DNA Polymerases and DNA Synthesis
In Vitro
The Complex Replication Apparatus
Unique Aspects of Eukaryotic
Chromosome replication
 Basic Features of DNA
Replication In Vivo
DNA replication occurs
semiconservatively, is initiated at
unique origins, and usually
proceeds bidirectionally from each
origin of replication.
DNA Replication is Semiconservative

Each strand serves

as a template
Complementary base
pairing determines
the sequence of the
new strand
Each strand of the
parental helix is
conserved
Possible Modes of
DNA Replication
The Meselson-Stahl Experiment:
DNA Replication in E. coli is Semiconservative
Semiconservative Replication
in Eukaryotes
Visualization of Replication in
E. coli (θ shape)
Replication in E. coli
The Origin of Replication in E. coli
.oriC -245 nt pairs long,two different
conserved repeat sequences
The Core Origin of Replication in SV 40
The Phage  Chromosome
Replication is Bidirectional
-AT rich molecules denatures more easily at low pH or
temperature.
Replication is Bidirectional
Denaturation Mapping
Bidirectional Replication of
Phage T7
 Key Points
 DNA replicates by a semiconservative mechanism:
as the two complementary strands of a parental
double helix unwind and separate, each serves as a
template for the synthesis of a new complementary
strand.

 The hydrogen-bonding potentials of the bases in the

template strands specify complementary base
sequences in the nascent DNA strands.

 Replication is initiated at unique origins and usually

proceeds bidirectionally from each origin.
DNA Polymerases and DNA
Synthesis In Vitro
Much of what we know about DNA
synthesis was deduced from in
vitro studies.
Requirements of DNA Polymerases

Primer DNA with

free 3'-OH
Template DNA to
specify the
sequence of the
new strand
Substrates: dNTPs
Mg2+
DNA Polymerase I
direction of synthesis is always 5’ to 3’
 DNA Polymerase I:
5'3' Polymerase Activity
 DNA Polymerase I:
5'3' Exonuclease Activity
 DNA Polymerase I:
3'5' Exonuclease Activity
 DNA Polymerases
Polymerases in E. coli
– DNA Replication: DNA Polymerases III and I
– DNA Repair: DNA Polymerases II, IV, and V
Polymerases in Eukaryotes
– Replication of Nuclear DNA: Polymerase 
and/or 
– Replication of Mitochondrial DNA: Polymerase 
– DNA Repair: Polymerases and
All of these enzymes synthesize DNA 5' to 3'
and require a free 3'-OH at the end of a primer
Phage T7 Polymerase
 DNA Polymerase III is the
True DNA Replicase of E. coli
E. coli DNA Polymerase III
Holoenzyme
Proofreading
living organisms have solved the potential problem of insufficient
fidelity during DNA replication
 Key Points
 DNA synthesis is catalyzed by enzymes called DNA
polymerases.

 All DNA polymerases require a primer strand, which

is extended, and a template strand, which is copied.

 All DNA polymerases have an absolute requirement

for a free 3'-OH on the primer strand, and all DNA
synthesis occurs in the 5' to 3' direction.
 Key Points
The 3'5' exonuclease activities of DNA
polymerases proofread nascent strands as
they are synthesized, removing any
mispaired nucleotides at the 3' termini of
primer strands.
 Key Points
 .DNA Polymerase 1 has two other exonuclease
activity: 5’ to 3’ and 3’ to 5’ other than the polymerase
activity. It is not the true ‘DNA replicase’ in E. coli
> DNA polymerase III is the DNA replicase responsible
for replication in E.coli. It is a multimeric enzyme

.
The Complex Replication
Apparatus
DNA replication is a complex
process, requiring the concerted
action of a large number of
proteins.
 DNA Replication
Synthesis of the leading strand is
continuous. Because the complementary strands of a
DNA double helix have opposite chemical polarity, one strand is
being extended in an overall 5’ to 3’ direction and the other
strand is being extended in overall 3’ to 5’direction(lagging
strand)

Synthesis of the lagging strand is

discontinuous. The new DNA is
synthesized in short segments (Okazaki
fragment) that are later joined together.
DNA Ligase Covalently
Closes Nicks in DNA
RNA Primers are Used to
Initiate DNA Synthesis
DNA Helicase Unwinds the
Parental Double Helix
Single-Strand DNA Binding
(SSB) Protein
Supercoiling of Unwound DNA
 DNA
Topoisomerase I
Produces Single-
Strand Breaks in
DNA
DNA Topoisomerase II Produces
Double-Strand Breaks in DNA
The Replication Apparatus in E. coli
Prepriming at oriC in E. coli
The E. coli Replisome
Rolling-Circle
Replication
 Key Points
DNA replication is complex, requiring the
participation of a large number of proteins.

DNA synthesis is continuous on the progeny

strand that is being extended in the overall
5'3' direction, but is discontinuous on the
strand growing in the overall 3'5' direction.
 Key Points
New DNA chains are initiated by short RNA
primers synthesized by DNA primase.

The enzymes and DNA-binding proteins

involved in replication assembled into a
replisome at each replication fork and act in
concert as the fork moves along the parental
DNA molecule.
Unique Aspects of Eukaryotic
Chromosome Replication
Although the main features of
DNA replication are the same in
all organisms, some processes
occur only in eukaryotes.
 DNA Replication in Eukaryotes
Shorter RNA primers and Okazaki
fragments
DNA replication only during S phase
Multiple origins of replication
Nucleosomes
Telomeres
Bidirectional Replication from
Multiple Origins in Eukaryotes
The Eukaryotic Replisome
 Eukaryotic Replication Proteins
 DNA polymerase -DNA  PCNA (proliferating cell
primase—initiation; nuclear antigen)—sliding
priming of Okazaki clamp
fragments  Replication factor-C Rf-C)
 DNA polymerase — —loading of PCNA
processive DNA  Ribonuclease H1 and
synthesis Ribonuclease FEN-1—
 DNA polymerase —DNA removal of RNA primers
replication and repair in
vivo
Disassembly and Assembly of
Nucleosomes
The Telomere Problem
Telomerase
Telomere Length and Aging
 Most human somatic
cells lack telomerase
activity.
 Shorter telomeres are
associated with cellular
senescence and death.
 Diseases causing
premature aging are
associated with short
telomeres.
 Key Points
 The large DNA molecules in eukaryotic chromosome
replicate bidirectionally from multiple origins.

 Two or three DNA polymerases (and/or ) are

present at each replication fork in eukaryotes.

 Telomeres, the unique sequences at the ends of

chromosomes, are added to chromosome by a
unique enzyme called telomerase.