Module 5a: Replicon

DNA replication: initiation, elongation & termination

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Learning objectives
By the end of this module, you should be able to:
1.Explain the role of DNA polymerases in synthesizing new DNA strands during replication.
2.Identify the various nuclease activities exhibited by DNA polymerases and their
significance in DNA replication.
3.Recognize the common structural features shared by different DNA polymerases.
4.Compare and contrast the modes of synthesis for the two new DNA strands during
replication.
5.Understand the importance of helicase and single-stranded binding proteins in the
replication process.
6.Analyze the coordination of lagging and leading strand synthesis during DNA replication.
7.Describe the role of the clamp in controlling the association of the core DNA polymerase
with DNA.
DNA Polymerases are the enzymes that make DNA
• DNA is synthesized in both semiconservative replication and DNA
repair reactions.
• DNA polymerase or DNA-dependent DNA polymerase

Figure 11.2: Semiconservative replication Figure 11.3: Repair synthesis replaces a short stretch
synthesizes two new strands of DNA. of one strand of DNA containing a damaged base.
DNA Polymerases are the enzymes that make DNA

Prokaryotic and eukaryotic:

• DNA polymerases have synthetic activity
from a 5' to 3' direction, using a template that
runs in the 3' to 5' direction.

• This process involves the incremental

addition of nucleotides to the growing strand,
one at a time, at a 3'-OH terminus.

• The precursor for DNA synthesis is a

nucleoside triphosphate

Figure 11.4: DNA is synthesized by adding

nucleotides to the 3ʹ –OH end of the growing chain.
DNA Polymerases are the enzymes that make DNA

A bacterium or eukaryotic cell has several different DNA

polymerase enzymes.
Bacteria
• DNA polymerase I (encoded by polA): Involved in repairing
damaged DNA and has a subsidiary role in semiconservative
replication.
• DNA polymerase II: Required for restarting a replication fork
blocked by DNA damage.
• DNA polymerase III: Responsible for de novo synthesis of
new DNA strands during replication.
• DNA polymerases IV and V: Involved in allowing replication
to bypass certain types of damage and are referred to as
error-prone polymerases.

Table 11.1: Only one DNA polymerase is the replication

enzyme.
DNA Polymerases are the enzymes that make DNA
A bacterium or eukaryotic cell has several different DNA polymerase enzymes.
Eukaryotic:
•DNA polymerases δ and ε are required for nuclear replication.
•DNA polymerase α is involved in "priming" or initiating replication.
•Other DNA polymerases play roles in repairing damaged nuclear DNA.
•Some DNA polymerases are involved in translesion replication of damaged DNA when
repair is impossible.
•Mitochondrial DNA replication is carried out by DNA polymerase γ.
•Chloroplasts have their own replication system.
DNA Polymerases have various nuclease activities

• Some have nuclease activity

• DNA polymerases provide a "proofreading" error-control system
• A 3'–5' exonuclease activity is typically employed to remove bases
that have been incorrectly added to DNA.
• DNA Polymerase I
• involved in DNA repair
• removes the RNA primers at the 5′ end of each Okazaki fragment during replication
and replaces them with DNA
 DNA Polymerases have various nuclease activities
Polymerase I
• Large fragment: The Klenow fragment contains both
polymerase and 3′–5′ exonuclease (proofreading)
activities.
• Smaller fragment: has a 5′–3′ exonucleolytic activity,
capable of excising small groups of nucleotides (up
to about 10 bases).
• This exonucleolytic activity is coordinated with the
synthetic and proofreading activities.
• DNA polymerase I has a unique 5′–3′ exonuclease
activity that can be combined with DNA synthesis to
perform nick translation. Figure 11.5: Nick translation replaces part of a preexisting
strand of duplex DNA with newly synthesized material.
 DNA Polymerases have a common structure

Polymerase I: Klenow fragment

• Many DNA polymerases have a large cleft
composed of three domains that resemble a
right hand.
• The "palm" domain contains conserved sequence motifs
essential for the catalytic active site.
• The "fingers" play a role in correctly positioning the template
at the active site.
• The "thumb" binds DNA as it exits the enzyme and is crucial
for processivity.

• DNA lies across the “palm” in a groove

created by the “fingers” and “thumb.”

Figure 11.7: The structure of the Klenow fragment from

E. coli DNA polymerase I.
DNA Replication in Bacterial Cells
Semidiscontinuous Replication
▪ Both daughter strands are synthesized simultaneously.
▪ DNA polymerase molecules move along a template only
in a 3′ → 5′ direction.
• The leading strand is synthesized continuously.
• The lagging strand is synthesized discontinuously (as
Okazaki fragments)
▪ The two newly assembled DNA strands grow in opposite
directions, one growing toward the replication fork and
the other growing away from it.

The two strands of a double helix are

synthesized by different sequences of events.
Copyright ©2020 John Wiley & Sons, Inc.
The two new DNA strands have different nodes of synthesis

Figure 11.9: The leading strand is synthesized continuously, whereas the lagging
strand is synthesized discontinuously.
Replication Requires a Helicase and a Single-Stranded Binding Protein

Two types of functions are needed to convert

double-stranded DNA to the single-stranded
state:
• Helicase is an enzyme that separates (or
melts) the strands of DNA, often utilizing ATP
hydrolysis for energy.
• Single-stranded binding protein (SSB) binds
to the single-stranded DNA, protecting it and
preventing it from re-forming the duplex state.

Figure 11.10: A hexameric helicase

moves along one strand of DNA.
Priming Is Required to start DNA synthesis

• All DNA polymerases require a 3′–OH priming end to initiate DNA synthesis.
• Called: Primer

Figure 11.11: A DNA polymerase requires a 3ʹ –OH end to initiate replication.

Priming is required to start DNA synthesis

• The priming end can be provided by an RNA

primer, a nick in DNA, or a priming protein.
• For DNA replication, a special RNA polymerase
called a primase synthesizes an RNA chain that
provides the priming end.
• E. coli: DnaG primase

Figure 11.12: There are several methods for providing the free 3ʹ –OH end that DNA
polymerases require to initiate DNA synthesis.
 • Priming of replication on double-
stranded DNA always requires a
Helicase, SSB, and primase.
• DnaB is the helicase that unwinds DNA for
replication in E. coli.

Figure 11.13: Initiation requires several enzymatic activities, including

helicases, single-strand binding proteins, and synthesis of the primer.
Coordinating synthesis of the lagging and leading Strands

Figure 11.14: A replication complex contains separate catalytic units for synthesizing the
leading and lagging strands.
The structure and functions of DNA Polymerase

▪ DNA polymerase III holoenzyme contains various

subunits having different functions in the replication
process.
▪ The DNA Pol III holoenzyme has at least two catalytic
cores, a processivity clamp, and a dimerization
clamp-loader complex.
DNA polymerase III Holoenzyme

• A clamp loader places the processivity subunits on DNA,

where they form a circular clamp around the nucleic acid.
• At least one catalytic core is associated with each template
strand.
• The E. coli replisome is composed of the holoenzyme
complex and the additional enzymes required for DNA
replication.

Figure 11.15: DNA polymerase III holoenzyme assembles in stages, generating an

enzyme complex that synthesizes the DNA of both new strands.
The clamp controls association of core enzyme with DNA
• A catalytic core is associated with each template
strand of DNA.
• The holoenzyme moves continuously along the
template for the leading strand.
• For the lagging strand, the template is "pulled
through," resulting in a loop in the DNA.
• DnaB plays a role in creating the unwinding point
and moves forward along the DNA

Figure 11.17: The helicase creating the

replication fork is connected to two DNA
polymerase catalytic subunits.
The clamp controls association of core enzyme with DNA
• The core on the leading strand is processive because
its clamp keeps it on the DNA.
• The clamp associated with the core on the lagging
strand dissociates at the end of each Okazaki
fragment and reassembles for the next fragment.

Figure 11.17: The helicase creating the replication fork is

connected to two DNA polymerase catalytic subunits.
The structure and functions of DNA Polymerase
DNA polymerase III holoenzyme
▪ b clamp keeps polymerase associated with DNA
▪ After the completion of one Okazaki fragment,
the enzyme disengages from its β clamp and
cycles to a recently assembled clamp “waiting” at
an upstream RNA primer–DNA template junction.
▪ Forms another Okazaki fragment
▪ The original β clamp is left behind for a period on
the finished Okazaki fragment, but it is eventually
disassembled and re-utilized.

Copyright ©2020 John Wiley & Sons, Inc.

The clamp controls the association of core enzyme with DNA

Figure 11.19: Core polymerase and the clamp dissociate at

completion of Okazaki fragment synthesis and reassociate at
the beginning.
The clamp controls the association of
core enzyme with DNA
• The helicase DnaB is responsible for
interacting with the primase DnaG to
initiate each Okazaki fragment.

Figure 11.18: Each catalytic core of Pol III synthesizes a daughter strand.
DnaB is responsible for forward movement at the replication fork.
Okazaki fragments are linked by ligase

• Each Okazaki fragment begins with a

primer and stops before the next
fragment.
• DNA polymerase I removes the primer
and replaces it with DNA.

Figure 11.20: Synthesis of Okazaki fragments

requires priming, extension, removal of RNA primer,
gap filling, and nick ligation.
Okazaki fragments are linked by ligase
• DNA ligase makes the bond that connects the 3′
end of one Okazaki fragment to the 5′ beginning
of the next fragment.
• enzyme–AMP intermediate

Figure 11.22: DNA ligase seals nicks between

adjacent nucleotides by employing an
enzyme-AMP intermediate.
DNA Replication in Bacterial Cells
Semidiscontinuous Replication

Copyright ©2020 John Wiley & Sons, Inc. Okazaki fragment synthesis at the lagging strand
 DNA Replication in Eukaryotic Cells
The Eukaryotic Replication Fork
▪ DNA is synthesized in a semidiscontinuous manner.
▪ Semiconservative and bidirectional
▪ Five polymerases: a, b, g, d e
• g replicates mtDNA Schematic view of the major components of
the eukaryotic replication fork. Proteins
• b - involved in DNA repair required for eukaryotic replication (left) and
• a - initiates Okazaki fragment synthesis replisome protein interactions (right).
• d - lagging strand synthesis
• e - leading strand synthesis

Copyright ©2020 John Wiley & Sons, Inc.

Termination of Replication

• The two replication forks usually meet

halfway around the circle, but there
are ter sites that cause termination if
the replication forks go too far.

Figure 11.27: Replication termini in E.

coli are located in a region between
two sets of ter sites.