DNA Replication Part I PDF

Mechanism of DNA replication
Subject : Molecular Biology
Lesson : Mechanism of DNA replication
Lesson Developer : Dr. Devi Lal
College / Department : Department of Zoology , Ramjas

College
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Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Mechanism of DNA replication
Topic Page No.
Introduction…………………………………………………………………………………………………………..1
Raw materials for DNA synthesis………………………………………………………….
Mechanism/model for DNA replication………………………………………………………….
DNA replication follows semi-conservative model………………………………
Enzymes involved in DNA replication………………………………………………………….

DNA polymerases………………………………………………………….…………………………………………
DNA polymerases in eukaryotes………………………………………………………….………………………
DNA polymerases in prokaryotes………………………………………………………….……………………..
Family of DNA polymerases………………………………………………………….……………………………..
Sliding clamps and clamp loader………………………………………………………….………
DNA ligase………………………………………………………….…………………………………………………
Primase………………………………………………………….………………………………………………………….
DNA helicases………………………………………………………….……………………………………………
Single stranded DNA binding protein (SSBs) …………………………………………
Topoisomerases………………………………………………………….……………………………………….
General principles of DNA replication…………………………………………………..
Summary………………………………………………………….…………………………………………………
Exercise/ Practice………………………………………………………….………………………………….
Glossary………………………………………………………….………………………………………………………
References/ Bibliography/ Further Reading………………………………………
Web links………………………………………………………….…………………………………………………
Answers………………………………………………………….……………………………………………………
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Introduction
The mid of nineteenth century was dominated by the work of Charles Darwin. Much
attention was focused on his work “On the Origin of Species”. The beginning of twentieth
century saw the rediscovery of Mendel’s law of heredity. Mid twentieth century was marked
by important discoveries that changed the perspective of molecular biologists. These
discoveries, that led to the origin of modern era in molecular biology were the landmark
works of Erwin Chargaff; Rosalind Franklin and Maurice Wilkins; James Watson and Francis
Crick. This work established the structure of DNA, the key macromolecule that directs life.
Once the structure of DNA was known, the scientists started looking for the mechanisms
that are responsible for duplication of this important molecule. The accurate replication of
DNA is important to pass correct genetic information from one generation to next. The
discovery that DNA replication is semi-conservative helped in understanding how DNA
replicates. Following this various enzymes were isolated that were known to play key role in
this process. Subsequently it was found that the process of DNA replication is same in
prokaryotes and eukaryotes employing same set of enzymes with some variation. In this
unit we will describe the functions of enzymes involved in DNA replication and basic
mechanism of this process.
Raw material for DNA synthesis
The synthesis of DNA requires certain raw materials. These are deoxyribonucleotides (dATP,
dCTP, dGTP and dTTP), primer, and the enzyme (DNA polymerase) that can faithfully copy
the template DNA. The primer is a short stretch of RNA that has the complementarity with
the template. A primer has a free 3’-OH that is extended by the DNA polymerase by
addition of the nucleotides. The addition of nucleotides takes place by formation of
phosphodiester bond. This reaction follows SN2 mechanism in which the exposed 3’-OH of
the primer attacks the α-phosphate group of the nucleotide, leaving pyrophosphate which is
subsequently hydrolyzed by pyrophosphatase. The hydrolysis of pyrophosphate provides the
energy for the reaction to proceed. The overall reaction is:
(NMP)n + NTP (NMP)n+1 + P—P 2Pi

Mononucleotide chain Incoming nucleotide Growing nucleotide pyrophosphate
triphosphate chain
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Mechanism/model for DNA replication
Three basic models have been proposed for DNA replication (Fig. 1). These are:
1) Conservative Model: This model assumes that the parent DNA strands serve as the
template for synthesis of complementary strand. After replication is finished, both
the parent strands are retained.
2) Semi-conservative Model: In this type of replication model, the parent DNA
strands separate and serve as a template for synthesis of new complementary
strand. Thus the daughter DNA produced after replication has one parental strand
and one newly formed strand. This is so far the correct model for DNA replication
which has been proved in the experiments of Meselson and Stahl.
3) Dispersive Model: In this model, the parent DNA strands are broken and serve as
template for the production of new DNA double helix.
Conservative
replication
Semi-conservative
replication
Dispersive
replication
Fig 1. Three models that were proposed to explain the possible ways in which the DNA
replicates. Out of these models semi-conservative replication model was supported from
the experimental evidences. (Source: Author)
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DNA replication follows semi-conservative model
Meselson and Stahl (1958) in a landmark discovery proved that DNA replication is semi-
conservative i.e. the two strands of duplex DNA separate and each strand serves as a
template for the production of complementary strand. The success of their experiment was
in part due to the use of density gradient centrifugation a technique in which molecules are
separated according to their densities. These workers allowed culture of E. coli to grow on a
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medium with heavy isotope of nitrogen N. Thus, E. coli grown in the presence of N would
incorporate this heavy nitrogen in their DNA making it denser (15N-15N). After growing in 15
N
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medium the bacteria were transferred to the medium with light isotope N and allowed to
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grow for two more generations. DNA was isolated from the culture grown in N medium
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and also from first and second generation grown on N medium. They subjected the
isolated DNA to cesium chloride density dependent centrifugation. The DNA isolated from
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the culture grown in N consisted of dense DNA which settled to the bottom of the
centrifuge tube (having salt solution of higher density) giving a single band. The DNA
isolated from first generation culture also gave a single band higher than the dense DNA
(because the density of this DNA was intermediate as it consisted of one light and one
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heavy strand, N-14N). The DNA from second generation yielded two bands of different
densities. One band was of the same density as that of first generation while the second
band was of low density (Fig. 2). This would have been possible only if DNA replication is
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semi-conservative. When the E. coli culture was transferred to N medium, after first round
of cell division the bacteria has incorporated light isotope of nitrogen in their DNA. So after
first generation, the bacteria has one DNA strand made of heavy isotope and other of light
isotope yielding a DNA of intermediate density (15N-14N). When this bacterial culture was
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allowed to grow for second generation in the same medium having N, the daughter DNA
would again incorporate the light isotope of nitrogen yielding two bands one with
intermediate density (15N-14N) and other of lighter density (14N-14N).
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E. coli transferred
to 14N medium
E. coli grown in E. coli grown in

15N medium 14N medium
Parent DNA First generation Second generation

(all heavy) (all intermediate) (half intermediate, half light)
15 N
14 N
Fig. 2. The experiment of Meselson and Stahl to prove semi-conservative replication of

DNA. (Source: Author)
Watch the animation of the experiment: <iframe width="420" height="315"

src="//www.youtube.com/embed/WsW2BZiLdpY" frameborder="0"
allowfullscreen></iframe>
Value addition: Video
6
Matthew Meselson (Harvard): The Semi-Conservative Replication of

DNA
Watch the talk by Meselson on Semi-conservative replication of DNA by clicking the

link below: https://www.youtube.com/watch?v=cvU4kEMvbm4
Source: iBioMagazine
Enzymes involved in DNA replication
DNA polymerases
DNA polymerases are the enzymes involved in DNA synthesis. DNA polymerases are
processive enzymes. The processivity of a DNA polymerase is the number of nucleotides
added by the polymerase each time it binds to the template. The processivity of DNA
polymerases vary with the type of polymerase and can range from few nucleotides to as
many as 50,000. The processivity of DNA polymerases is increased dramatically by their
association with the sliding clamps which are multi-subunit proteins (discussed below) and
prevent the dissociation of the polymerase from the template.
The initial studies on the DNA polymerase from E. coli linked the structure of the polymerse
with a right hand with the subdomains referred to as fingers, palm and thumb (Fig. 3). The
active site of polymerase resides in the palm subdomain which is composed on β-sheet and
binds two divalent metal ions like Mg2+ or Zn 2+
. The nucleotide recognition and binding is
carried out by finger subdomain while thumb subdomain is important for binding the
substrate DNA with polymerase. This general architecture is found to be conserved within
the polymerases despite little sequence homology.
Thumb subdomain
Finger subdomain
Palm subdomain
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Fig. 3. Structure of DNA polymerase resembles a right hand with specific role of each
subdomain; finger subdomain: nucleotide recognition and binding; thumb subdomain:
binding the substrate DNA with polymerase; palm subdomain: active site of polymerase.
(Source: Author)
DNA polymerases are specific enzymes and add a correct nucleotide to the growing DNA
chain. The accuracy of DNA polymerases depends on the ability of correct base pairing
between A-T and G-C base pair. The addition of wrong base lowers the rate of nucleotide
addition due to unfavorable alignment of the nucleotides. So DNA polymerases present an
example of kinetic selectivity where the correct bond formation takes place only in the
presence of correct nucleotide. Apart from this, DNA polymerases are efficient in
distinguishing between ribonucelotides and deoxyribonucleotides, by a phenomenon known
as steric exclusion. The active site in DNA polymerase is small enough to bind only
deoxyribonucleotides and cannot accommodate ribonucleotides which have extra O at 2’
position. Thus the overall activity of DNA polymerases depends on the geometry and
complementarity of base pair. Therefore, DNA polymerases can sometime tend to
incorporate wrong base in the growing nucleotide chain @ of 1 nucleotide per 1010
nucleotide added. The wrongly incorporated base is removed by the proofreading
exonuclease which is the part of DNA polymerase itself. It removes any wrong base from 3’
of the newly synthesized nucleotide chain. This proofreading activity is important as the
incorporation of wrong base can alter the genetic message that may prove lethal to the cell.
DNA polymerases in prokaryotes
Prokaryotes consist of five DNA polymerases (Table 1) of which only Pol III is involved in
DNA replication. The first polymerase to be identified was Pol I from E. coli. The enzyme
was isolated by Arthur Kornberg. The enzyme was found to have polymerase activity that
can extend a deoxyribonucleotide chain, 3’-5’exonuclease activity that can remove a
mismatched base and 5’-3’ exonuclease activity which degrades double stranded DNA. Pol I
is also involved in removal of RNA primers. Pol II is involved in DNA repair while pol III is
the main enzyme involved in DNA replication. Pol III is found to be a part of a complex
which forms a holoenzyme (Fig. 4). Each holoenzyme consists of two copies of DNA Pol III
core enzyme and a copy of clamp loader (γ-complex). Each DNA Pol III core enzyme
interacts with the γ-complex through τ protein. The τ protein consists of a flexible linker
which allows DNA Pol III core enzymes to move independently.
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Table 1: DNA polymerases identified in prokaryotes. Out of all these only DNA pol III
is the chief enzyme involved in replication of bacterial chromosome.
S. No. Name of polymerase Family Function

1. Pol I A Excicion repair, removal of RNA primers
2. Pol II B DNA repair
3. Pol III C DNA replication
4. Pol IV Y DNA repair
5. Pol V Y DNA repair and translesion synthesis
τ-subunit
DNA pol III DNA pol III
core enzyme core enzyme
Clamp loader
(γ-complex)
Sliding clamp
Fig. 4. The structure of bacterial DNA pol III holoenzyme.

(Source: Author)
DNA polymerases in eukaryotes
Eukaryotes consist of large number of DNA polymerases as compared to prokaryotes (Table

2). Out of these polymerases atleast three polymerases α, δ and ε are needed for DNA
replication, while others seem to be needed for repair of damaged DNA. DNA pol α
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holoenzyme has both primase and polymerase activity and is involved in the primer
synthesis during DNA replication. The polymerase subunit extends the primer synthesized
by primase subunit. After DNA pol α has extended the primer to some 30-40 nucleotide
polymerase switching takes place. In polymerase switching pol α is displaced from the
template and the synthesis is taken over by pol δ and ε. The polymerase switching is
required as pol α lacks ′3 -5′ exonuclease activity and has low processivity. While pol δ is
involved in synthesis of lagging strand, pol ε does the leading strand synthesis.
Value addition: Interesting to know
Contributions of Arthur Korenberg
Arthur Kornberg is credited for the discovery of DNA polymerase from E. coli in the
year 1957. He along with his coworkers isolated an enzyme and used it to direct DNA
synthesis in an in vitro system. His early studies were based on the enzymatic
synthesis of coenzymes. Later on he became interested in the synthesis of nucleic
acids. He could elucidate key steps in the synthesis of purine and pyrimidines. He
also showed that the synthesis of DNA takes place only in a single direction.
However, later it was found that the Kornberg enzyme or Polymerase I was is not the
chief enzyme involved in DNA replication. This led to the subsequent discovery of
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other DNA polymerases from E. coli and could help in understanding the mechanism
of DNA replication. He was awarded the Nobel prize (1959) in physiology and
medicine.
Source:
http://upload.wikimedia.org/wikipedia/commons/b/b5/Arthur_Kornberg_1969_B.png
Table 2: DNA polymerases found in eukaryotes.
DNA pol α, δ and ε are involved in replication of chromosomal DNA.
S. No. Name of Family Proposed function

polymerase
1. α B DNA replication
2. β X Base excision repair
3. γ A Mitochondrial replication
4. δ B DNA replication, Nucleotide-excision
repair, Base-excision repair, Mismatch
repair, Double-strand break repair
5. ε B DNA replication, Nucleotide-excision
repair, Base-excison repair, Mismatch
repair, Double-strand break repair
6. ζ B Translesion synthesis
7. η Y Translesion synthesis
8. θ A DNA repair
9. ι Y Translesion synthesis
10. κ Y Translesion synthesis
11. λ X Base excision repair
12. μ X Non homologous end joining
13. σ X Sister chromatid cohesion
14. Rev1 Y Translesion synthesis
15. Telomerase RT Telomere maintenance
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Family of DNA polymerases
The DNA polymerases have been classified into 7 families (Table 3). These classes have
been denoted by letter A, B, C, D, X, Y and RT (reverse transcriptase).
Family A: The prototype enzyme of this family is pol I. Though polI has minor role in
replication other member of this family like T7 DNA polymerase is involved in DNA
replication of phage T7. The members of this family have
′ 3 -5′ exonuclease activity
(proofreading activity) which is conserved.
Family B: This family consists of main replicative enzymes of eukaryotes which are involved
in replicating large DNAs of eukaryotes. Family B polymerases are mainly multi subunit
enzymes. The members of this family also have′ 3 -5′ exonuclease activity (proofreading
activity).
Family C: This family consist of main replicative enzyme of prokaryotes i.e. pol III.
Family D: The family D polymerases are exclusive to the archaea. These polymerases are
two-subunit enzymes comprised of DP1 and DP2. The polymerase activity is found to be
present within subunit DP2. The members of this family have 3′-to-5′ exonuclease activity.
Family X: Polymerases belonging to this family are small and monomeric like DNA pol λ,
DNA pol μ etc. These polymerases are mainly involved in DNA repair.
Family Y: The members belonging to this family do not have any exonuclease activity.
These polymerases are mainly involved in Translesion synthesis.
Table 3: The family of DNA polymerases.
The DNA polymerases from all living organisms have been divided into 7 families.
S. No. Class Example

1. A Pol I, DNA pol γ, T7 DNA polymerase
2. B Pol II, DNA pol α, DNA pol ε
3. C Pol III,
4. D polymerases exclusive to archaea
5. X DNA pol β, DNA pol λ, DNA pol μ, DNA pol σ
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6. Y Pol IV, polV, DNA pol η, DNA pol ι, DNA pol κ

7. RT Telomerase
Proofreading of DNA polymerase
Watch the talk animation on proofreading ability of DNA polymerae by clicking the
link below:
https://www.youtube.com/watch?v=LvS_Gwb2wY8
Source: You Tube
Sliding clamps and clamp loader
As already mentioned, the processivity of DNA polymerase is increased with their

association with the sliding clamps. These multisubunit ring-like proteins are found to be
highly conserved and share a similar sixfold symmetry. In prokaryotes these are known as
beta subunit while in eukaryotes these are known as PCNA (Proliferating cell nuclear
antigen). These proteins encircle the double helix of DNA and slide along the DNA with the
polymerase without getting dissociated from it. The sliding clamps prevent the diffusion of
the polymerase once it gets dissociated from the template. Thus, the sliding clamps ensure
the close proximity between DNA and the polymerase increasing the processivity of the
enzyme. Once the complementary strand has been synthesized, there is decrease in the
affinity of sliding clamp with the polymerase. This results in the release of the polymerase
from the DNA and the sliding clamp. The release of polymerase is not immediately followed
by the release of sliding clamp. Rather various other proteins and factors become associated
with the sliding clamp after the release of the polymerase. These include the proteins
involved in repair of okazaki fragments and chromatin assembly.
Sliding clamp loaders are protein complexes that are required for loading as well as
unloading the sliding clamps on/from the DNA using energy from ATP hydrolysis (Fig. 5).
When bound with ATP, clamp loaders bind with the sliding clamp resulting in opening of the
ring of the sliding clamp. The opened ring sliding clamp is loaded onto the DNA: primer
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junction. This binding results in the hydrolysis of ATP resulting in the release of clamp
loader. The sliding clamps are removed only when all the proteins and factors that act on
newly synthesized DNA have interacted with the sliding clamp. Clamp loaders are known as
γ-complex in prokaryotes and RF-C (Replication Factor C) in eukaryotes.
ATP ATP ATP
Clamp loader (γ-

complex) Sliding clamp
(C)
(A) (B)
ATP
ATP hydrolysis (D)
ADP
Pi
(E)
Fig. 5. Sliding clamp loading by the clamp loader is controlled by ATP hydrolysis. (A) The
clamp loader is known as γ-complex in E. coli and has five subunits. (B) The clamp loader
binds a molecule of ATP. (C) Once the clamp loader has ATP molecule bound to it, it binds
with the sliding clamp which results in the opening of the ring of the clamp. (D) This
complex binds with the DNA resulting in the hydrolysis of ATP to ADP. (E) This results in
dissociation of clamp loader from the sliding clamp, and simultaneous binding of DNA
polymerase. (Source: Author)
DNA ligase
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DNA ligase is involved in closing the nicks in the phosphodiester backbone of DNA and
therefore is essential for joining the Okazaki fragments. These are also involved in sealing
the nicks after DNA damage repair. There are two main classes of DNA ligases: one which
uses NAD+ as cofactor and is present in prokaryotes and the second which uses ATP as
cofactor and is present in eukaryotes and viruses. Those ligases that use ATP as cofactor
are of four types:
• DNA ligase I: links Okazaki fragments.
• DNA ligase II: an alternative spliced form of ligase III, mainly found in non dividing
cells.
• DNA ligase III: involved in base excision repair.
• DNA ligase IV: involved in repair of double stranded DNA breaks
Primase
Primase is the enzyme involved in the synthesis of short stretch of RNA that is extended by
DNA polymerase. DNA polymerase cannot start the synthesis of DNA de novo, it can only
extend the 3’ end of an already exisiting nucleotide chain. Therefore, the role of primase is
important in DNA replication. Since the primase synthesizes RNA primer, it is a type of RNA
polymerase. Leading strand requires a single primer while lagging strand requires a number
of primers to be synthesized, one per Okazaki fragment. Primase is also known as DnaG in
prokaryotes and contains N-terminal Zn binding domain, C-terminal DnaB (DNA helicase)
binding domain and a central domain that has the primase activity.
DNA helicases
DNA helicases are hexameric enzymes that are involved in separation of two strands of a
DNA duplex. These enzymes use energy from ATP hydrolysis to move along the DNA in a
particular direction. This property is known as polarity. So helicases have definite polarity
which can be either 3’-5’ or 5’-3’. DNA helicases also act processively like DNA
polymerases. In prokaryotes they are known as DnaB while in eukaryotes Mcm 2-7 complex
(minichromosome maintenance). It has been found that DnaB helicase shares significant
sequence similarity with RecA protein (required for DNA repair and recombination). Mcm 2-7
complex initially identified in budding yeast consists of 6 unique subunits and is found to be
highly conserved across the eukaryotes.
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Single stranded DNA binding protein (SSBs)
DNA helicases are responsible for unwinding the double helix of DNA so that the polymerase
can copy the genetic information to synthesize the complementary strand. Single stranded
DNA binding proteins bind with the single strand generated by the action of the helicases to
prevent the reformation of the double strand. They do this in sequence independent
manner. Apart from their role in replication SSBs are also involved in DNA repair and
recombination. The binding of one SSB to the ssDNA promotes the binding of the other
SSBs. This phenomenon is known as cooperativity. Eukaryotic counterpart of SSB is known
as RPA (Replication protein A).
Topoisomerases
These are the enzymes that are involved in interconversions of various topological forms of
DNA. In DNA replication these enzymes are responsible for removing the supercoils
introduced by the action of helicases. Topoisomerases relax the supercoils by breaking one
or both the strands of DNA. Based on number of strands broken, topoisomeraes have been
grouped into two classes: (i) Topoisomerase I and (ii) Topoisomerase II. The first
topoisomerase discovered was from E. coli now known as topoisomerase I. Topoismerases
belonging to Class I cleave only one strand of DNA. It acts on negative supercoils but fails
to act on positive supercoils. Type II topoisomerases cleave both the strands of DNA. First
type II topoisomerase was also isolated from E. coli and was known as DNA gyrase. Class II
topoisomeraes use energy from ATP hydrolysis to break and rejoin the DNA strands while
Class I topoisomerases do not use ATP as the reaction is energetically favorable.

Role of Topoisosermase in solving knotty problem of DNA!!!!
Watch the animation and learn about topoisomerase
https://www.youtube.com/watch?v=EYGrElVyHnU
Source: You Tube
General principles of DNA replication
The replication of DNA always starts at the discrete sites known as origin of replication.
The origin of replication serves as the site for binding of a number of proteins that can
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initiate the process of replication. The origin of replication from E. coli was the first origin of
replication to be defined and studied in detail. Since then, origin of replication from a
number of organisms including viruses and animals has been characterized. The
prokaryotes because of small genome size have usually a single origin of replication. In case
of E. coli, the origin of replication is known as oriC, which is about 245 base pair and
consists of AT rich 13 mer repeats. The entire E. coli genome (4X106 bp) is replicated from
this origin in approximately 30 min! The bacterial counterparts archaea also have small
genome size and have usually single origin but multiple origins have been reported in a
number of archaea like Sulfolobus solfataricus and Sulfolobus acidocaldarius. Moreover, the
replication machinery of archaea also bears remarkable resemblance with the eukaryotes.
Like bacteria, viruses also have a single origin of replication e.g. SV40 virus has 64 base
pair single origin of replication. SV40 virus encoded protein T antigen binds with the origin
and initiates the process of replication. Eukaryotes because of huge genome size require
multiple origins to replicate their DNA. These multiple origins are present about 50-300 kb
apart. In case of yeast, the origin was identified as the sequences that can support the
replication of plasmids and were named as autonomously replicating sequences
(ARSs). The origins from all eukaryotes form an assembly before the replication known as
pre-RC or pre replication complex.
In order to synthesize a complimentary copy of DNA the two strands of double helix should
separate, forming two template strands. This region where the two stands separate forms a
replication fork. The replication forks always move towards the unreplicated duplex DNA,
leaving behind two template strands. The replication fork is created by the action of DNA
helicases. The bacterial chromosomes are circular and therefore the unwinding of DNA at
origin leads to the formation of two replication forks. The replication of such circular
chromosomes proceeds in opposite direction to the terminus. Such process is termed as bi-
directional replication (Fig. 6).
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OriC Replication fork Replication fork
Fig. 6. The bidirectional replication of circular chromosome of E. coli which proceeds with
two replication forks. (Source: Author)
The single stranded DNA template created by the action of DNA helicases is rapidly bound
by the single stranded DNA binding proteins (SSBs) to prevent the reformation of duplex
DNA. Now the primase starts synthesizing short primer strands that are extended by the
DNA polymerase. DNA polymerase can extend an already existing DNA chain by adding
nucleotides to 3’ end, and two DNA chains are antiparallel in nature. Because of this only
one of the exposed templates is replicated continuously. The DNA synthesized using this
template is known as the leading strand and the other strand that is produced in
discontinuous manner is known as the lagging strand. The overall process is referred to as
semi-discontinuous DNA replication. The evidence for this semi-discontinuous DNA
replication was provided by Okazaki (1968). The leading strand is thus synthesized in the
direction of growing replication fork while the lagging stand synthesis takes place in the
opposite direction of the growing replication fork (Fig. 7). Therefore, the synthesis of
lagging strand is more complicated than leading strand synthesis and takes place only when
a considerable length of the lagging strand template is exposed. The lagging strand requires
synthesis of several short RNA primers that are extended by the DNA polymerase to
produce Okazaki fragments. The Okazaki fragments vary in length from 1000-2000
nucleotides in bacteria and 100-400 nucleotides in eukaryotes. Once the synthesis of
Okazaki fragments is complete, the RNA primers are removed. In E. coli, these RNA primers
are removed by the action of DNA polymerase I which has exonuclease activity (in either 3’-
5’ or 5’—3’). In case of eukaryotes the RNA primers are removed by the action of RNase H
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which acts on RNA-DNA hybrid strands. The resulting gap is then filled by DNA polymerase
and the nick left is joined by DNA ligase.
Leading strand DNA

5’ synthesis
3’
DNA polymerase III Topoisomerase II
3’
τ-subunit 5’
Sliding clamp
Helicase Primase
5’ Single stranded DNA
Primer
3’ binding protein
5’ Lagging strand DNA DNA polymerase III

synthesis 5’
Fig. 7. The figure depicts the replication fork created by the action of helicase. Because of
the antiparallel nature of DNA double helix, one strand is continuously synthesized (known
as leading strand) and the synthesis of the other strand takes place in discontinuous
manner (lagging strand). (Source: Author)
Value addition: Interesting to know!!!!
Reiji Okazaki
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Reiji Okazaki was born in Hiroshima in 1930. He is

known for his significant contributions in
understanding the mechanism of DNA replication. He
along with his wife Tsuneko designed experiments in
E. coli to understand the replication of a DNA
molecule. They used pulse-labeling technique in which
growing E. coli culture was exposed to brief pulse of
Value addition: Video radioactive thymidine. They isolated the DNA and
denature it in an alkaline solution and separate the
DNA Replication:
DNA using Okazaki
centrifugation. They fragments
could find several short DNA fragments which were
an evidence of dis-continuous DNA replication. These short DNA fragments are now
Watch the animation and learn about Okazaki fragments
known as Okazaki fragments. As a child Okazaki was exposed to the radiation
https://www.youtube.com/watch?v=OXb_kKyKgwM
because of atomic bomb explosion in Hiroshima. He died at the age of 44 because of
leukemia.
Source: You Tube
Source:http://ap-biology-lab.wikispaces.com/file/view/192px-
Okazaki_Reiji.jpg/176168959/192px-Okazaki_Reiji.jpg
leading strand and lagging strand
Watch the animation and learn about the replication of leading strand and lagging
strand
https://www.youtube.com/watch?v=t43YVaBagOI
Source: You Tube
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Summary
• DNA replication is the process of making the identical copy of DNA.
• DNA replication requires raw materials like deoxyribonucleotides (dATP, dCTP, dGTP
and dTTP), primer, and DNA polymerase.
• A primer provides free 3’-OH that is extended by the DNA polymerase by addition of
the nucleotides which follows SN2 reaction mechanism.
• Three models of DNA replication have been proposed conservative, semi-

conservative and dispersive model.
• Out of these possible models, conservative model gain evidences from the
experiments of Meselson and Stahl and thought to represent the actual mode of DNA
replication.
• Inside a cell, a number of enzymes and proteins are involved in replicating DNA. DNA
polymerases are the chief enzymes that copy a template DNA to produce its
complementary strand. DNA polymerases are processive enzymes with the structure
resembling the right hand.
• DNA polymerases work using the phenomenon of kinetic selectivity and steric
exclusion. Prokaryotes consist of five DNA polymerases of which only Pol III is
involved in DNA replication. Pol III is found to be a part of a complex which forms a
holoenzyme having two copies of DNA Pol III core enzyme and a copy of clamp
loader (γ-complex).
• In eukaryotes three polymerases α, δ and ε are needed for DNA replication. DNA pol
α holoenzyme has both primase and polymerase activity and is involved in the
primer synthesis during DNA replication.
• Pol δ and pol ε areis involved in synthesis of lagging strand and leading strand
respectively.
• The DNA polymerases have been classified into 7 families denoted by letter A, B, C,
D, X, Y and RT (reverse transcriptase). Out of which family C consist of main
replicative enzyme of prokaryotes.
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• Sliding clamps are multisubunit ring-like proteins which encircle the double helix of
DNA and slide along the DNA with the polymerase increasing the processivity of the
enzyme. These are known as beta subunit in prokaryotes and PCNA (Proliferating cell
nuclear antigen) in eukaryotes.
• Sliding clamp loaders are protein complexes that are required for loading as well as
unloading the sliding clamps on/from the DNA using energy from ATP hydrolysis.
Clamp loaders are known as γ-complex in prokaryotes and RF-C (Replication Factor
C) in eukaryotes.
• Other enzymes involved in replication are DNA helicases that are involved in
separation of two strands of a DNA duplex, primase involved in the synthesis of short
stretch of RNA that is extended by DNA polymerase, ligase involved in closing the
nicks in the phosphodiester backbone of DNA, and topoisomerases are involved
removing the supercoils introduced by the action of helicases.
• Apart from these enzymes a group of proteins, single stranded DNA binding proteins
bind with the single strand generated by the action of the helicases to prevent the
reformation of the double strand.
• The origin of replication is discrete site that bind a number of proteins that can
initiate the process of replication.
• Prokaryotes generally have a single origin while eukaryotes because of huge genome
size require multiple origins to replicate their DNA. Multiple origins have been
reported in a number of archaea.
• In case of E. coli, the origin of replication is known as oriC and it is known as

autonomously replicating sequences (ARSs) in yeast. The region where the two
stands of double helix separate forming two template strands forms a replication
fork. The prokaryotes have two replication forks because of circular chromosomes
and therefore the replication proceeds in opposite direction to the terminus. Such
process is termed as bi-directional replication.
• During replication only one of the strands is synthesized continuously and is known
as leading strand and the other strand that is produced in discontinuous manner is
known as the lagging strand.
22
• The overall process is referred to as semi-discontinuous DNA replication. Therefore

the synthesis of lagging strand requires several short RNA primers that are extended
by the DNA polymerase to produce Okazaki fragments.
• Once the synthesis of Okazaki fragments is complete, the RNA primers are removed.
In E. coli, these RNA primers are removed by the action of DNA polymerase I while
in eukaryotes theyare removed by the action of RNase.
Exercise/ Practice
A. Multiple choice questions:
1. Which of the following enzymes are used to join bits of DNA?
(a) DNA ligase (b) DNA polymerase (c) primase (d) Endonuclease
2. Each strand of the DNA molecule can be used to make another strand during
replication. This is because the two sides are ______ to each other.
(a) homologous (b) complimentary (c) identical (d) translatable
3. Topoisomerase enzymes are important in the replication of DNA because they
(a) anneal Okazaki fragments (b) "proofread" newly synthesized DNA
(c) degrade histone proteins (d) relax supercoiled DNA
4. DNA replication
(a) is not edited once polymerization has occurred (b) is conservative.
(c) requires a type of RNA polymerase (d) is partially regulated by promoter/terminator

sites.
5. Proofreading is performed by the _______________________ (activity) of DNA

polymerase?
(a) ligase (b) exonuclease (c) polymerase (d) kinase
6. The lagging strand is synthesized discontinuously at the replication fork because
(a) the lagging strand template is discontinuous.
23
(b) DNA polymerase always falls off the template DNA every ten nucleotides
(c) DNA polymerase removes the last few nucleotides synthesized whenever it stops.
(d) DNA polymerase can polymerize nucleotides only in the 5'-to-3' direction.
7. In the DNA polymerase reaction, incoming nucleotides are covalently bonded to the
___________ end of the growing DNA chain. Each successive nucleotide is linked to the
growing chain by a _______________ bond.
(a) 5', hydrogen (b) 5', phosphoester (c) 3', hydrogen (d) 3', phosphoester
8. DNA polymerase γ is responsible for the
(a) Removal of RNA primers (b) Elongation of the leading strand
(c) Elongation of the lagging strand (d) Replication of mitochondrial DNA
9. What is the primary function of primosomes?
(a) To add the 5'-methyl cap to pre-mRNA (b) To prevent binding of the ribosome to the
mRNA molecule (c) To initiate the process of translation (d) To create RNA primers for
DNA replication
10. the polarity of DNA synthesis is
(a) 5’-----3’ (b) 3’------5’ (c) 5’------2’ (d) 2’------5’
11. Mode of DNA replication in E.coli is
(a) Conservative and unidirectional (b) Semiconservative and unidirectional

(c) Conservative and bidirectional (d) Semiconservative and bidirectional
12. DNA synthesis can be specifically measured by estimating the incorporation of radio
labelled
(a) uracil (b) thymine (c) Adenine (d) Deoxyribose sugar
13. During the replication of DNA, the synthesis of DNA on lagging strand takes place in
segments, these segments are called
(a) Satellite segments (b) Double helix segments (c) Kornbeg segments (d) Okazaki
segments
24
14. Which of the following polymerases is not involved in replication in eukaryotes:

(a) α (b) β (c) ε (d) δ
15. The first polymerase to be discovered was

(a) Pol I (b) Pol III (c) pol α (d) pol δ
B. Fill in the blanks:
1. The _______________ strand is synthesized continuously during DNA replication.
2. The lagging strand consists of ______________ fragments that are later on joined by
_______________ to form one strand of DNA.
3. DNA replication occurs in ___________ phase of cell cycle.
4. The _______________ of a DNA polymerase is the number of nucleotides added by the

polymerase each time it binds to the template.
5. DNA pol _____________ has both primase and polymerase activity.
6. DNA polymerases can distinguish between ribonucelotides and deoxyribonucleotides, by a

phenomenon known as ___________________.
7. Enzymes that are involved in separation of two strands of a DNA duplex are
____________________.
8. Single Stranded DNA Binding protein of eukaryotes is known as ______________.
9. ____________ topoisomerases cleave both the strands of DNA.
10. In DNA pol III holoenzyme each DNA Pol III core enzyme interacts with the γ-complex
through ________________.
11. The replication of DNA always starts at ______________.
12. The origin of replication in yeast was identified as ____________________.
13. In prokaryotes the RNA primers are removed ________________ while in eukaryotes
they are removed by ________________.
25
14. The activity DNA polymerases depends on the ability of correct base pairing between A-
T and G-C base pair rather than identifying correct base. This presents an example of
_______________.
15. Model of replication explained by Meselson and Stahl is _______________________.
C. True/False
1. A phosphodiester bond joins one nucleotide together in the DNA polymer.
2. DNA topoisomerase supercoils DNA to get it out of the way of DNA polymerase.
3. The active site of polymerase resides in the palm subdomain.
4. The nucleotide recognition and binding is carried out thumb subdomain of DNA
polymearse.
5. Single stranded DNA binding proteins show cooperativity.
6. Unlike bacteria archea have multiple origin of replication.
7. Archea resemble bacteria in mechanism and machinery for replication as both belong to
the kingdom Monera.
8. Family C of DNA polymerases consists of main replicative enzyme of prokaryotes.
9. DNA ligase II links Okazaki fragments.
10. Okazaki discovered the first DNA polymerase from E. coli.
D. Expand the following
1. RT; 2. PCNA; 3. RF-C; 4. Mcm complex; 5. SSBs; 6. RPA; 7. ARSs; 8. pre-RC
E. Short answer questions

1. During the DNA replication process, RNA primers are removed and replaced with DNA.
Explain why it is important to replace RNA with DNA.
26
2. When Meselson and Stahl showed that replication is a semiconservative process, using
isotopes of nitrogen (15N and 14N). Explain the expected results if replication would have
been conservative.
3. How will it be able to test if a particular DNA sequence acts like origin of replication in
yeast?
4. How many Okazaki Fragments would be expected to synthesized in replicating an E. coli
genome with genome size 4X106?
5. What defects would be expected in an E. coli strain if it has a mutant DNA pol I?
Glossary
autonomously replicating sequences: DNA sequences in yeast that serve as origin of
replication
bi-directional replication: replication of circular chromosomes leads to formation of two

replication forks and replication proceeds in opposite direction to the terminus
Cooperativity: The phenomenon in which binding of one ligand molecule to the

macromolecule promotes the binding of the other ligands
DNA polymerases: enzymes involved in DNA synthesis
helicases: hexameric enzymes involved in separation of two strands of a DNA duplex
lagging strand: During DNA replication the strand that is produced in discontinuous
manner
leading strand: During DNA replication the strand that is replicated continuously
ligase: enzymes involved in closing the nicks in the phosphodiester backbone of DNA
Okazaki fragments: fragments that are produced in lagging strand synthesis
origin of replication: discrete sites from where the replication of DNA starts
Polarity: The property of helicases to move along the DNA in a particular direction using
energy from ATP hydrolysis
Primase: enzyme involved in the synthesis of short stretch of RNA that is extended by DNA
polymerase
27
Processivity: number of nucleotides added by the polymerase each time it binds to the
template
semi-discontinuous DNA replication: replication that proceeds with the synthesis of

leading (strand that is synthesized continuously) and lagging strands(strand that is
synthesized discontinuously)
Single stranded DNA binding proteins: These bind with the single strand generated by
the action of the helicases to prevent the reformation of the double strand.
Topoisomerases: enzymes responsible for removing the supercoils introduced by the

action of helicases
References/ Bibliography/ Further Reading

Burgers, P. M. 1998. Eukaryotic DNA polymerases in DNA replication and DNA repair.
Chromosoma 107:218-27.
Caruthers, J.M. and McKay, D. B. 2002. Helicase structure and mechanism. Curr. Opin.
Struct. Biol. 12: 123-33.
Champoux, J. J. 2001. DNA topoisomerases: structure, function, and mechanism. Annu.

Rev. Biochem. 70: 369-413.
Cooper, G. M. and Hausman, R. E. 2007. Replication, maintenance and rearrangements of

genomic DNA. In The cell: a molecular approach. 4th eEdition. ASM Press.
Hübscher, U. 1983. DNA polymerases in prokaryotes and eukaryotes: Mode of action and
biological implications. Experientia 39: 1-25.
Jeruzalmi, D., O'Donnell, M. and Kuriyan, J. 2002. Clamp loaders and sliding clamps. Curr.
Opin. Struct. Biol. 12: 217-24.
Kelman, Z. and O'Donnell, M. 1995. DNA POLYMERASE III HOLOENZYME: structure and
function of a chromosomal replicating machine. Annu. Rev. Biochem. 64: 171-200.
Klug, W. S., Cummings, M. R. and Spencer, C. A. 2006. DNA replication and recombination.
In concepts of genetics. 8th edition. Pearson education international.
28
Kornberg, A. and Baker T. 1992. DNA Replication. 2nd Edition, W.H. Freeman and Company,
New York.
Lehman, I.R. 1974. DNA ligase: structure, mechanism, and function. Science 186: 790-
797.
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. Nat. Acad. Sci. USA 59: 598-605.
Steitz, T. A. 1999. DNA polymerases: structural diversity and common mechanisms. J.

Biological Chem. 274: 17395-17398.
Watson, J.D., Baker, T. A. , Bell, S.P., Gann, A., Levine, M., and Losick, R. 2013. The
replication of DNA. In Molecular Biology of the Gene. 7th edition. Pearson education
international.
Web links
• http://www.cliffsnotes.com/sciences/biology/biochemistry-ii/dna-structure-
replication-and-repair/dna-replication-enzymes
• https://www.youtube.com/watch?v=27TxKoFU2Nw
• https://www.youtube.com/watch?v=2iVltkYy0jg
• http://www.youtube.com/watch?v=JcUQ_TZCG0w
Answers
A. Multiple choice questions:

1. (a) 2. (b) 3. (d) 4. (c) 5. (b) 6. (d) 7. (d) 8. (d) 9. (d) 10. (a) 11. (d) 12. (b) 13. (d)
14. (b) 15. (a)
B. Fill in the blanks:

1. leading 2. Okazaki, Ligase 3. S 4. Processivity 5. α 6. steric exclusion
29
7. DNA helicases 8. Replication protein A 9. Type II 10. τ protein 11. origin of replication
12. autonomously replicating sequences (ARSs) 13. polymerase I, RNase H
14. kinetic selectivity 15. Semi-conservative.
C. True/False
1. True 2. False 3. True 4. False 5. True 6. True 7. False 8. True 9. False 10. False
D. Expand the following

1. reverse transcriptase 2. Proliferating cell nuclear antigen 3. Replication Factor C 4.
minichromosome maintenance complex 5. Single stranded DNA binding protein 6.
Replication protein A 7. autonomously replicating sequences 8. pre replication complex
E. Short answer questions

1. RNA is more unstable as compared to DNA. Therefore the RNA primers must be removed and
replaced with DNA which is more stable. Moreover, the replacement is also required to direct the next
round of replication.
2. If the replication would have been conservative than the first generation of replication would yield 2
bands -15N and 14N and no hybrids as obtained by Meselson and Stahl. Similarly results would have
seen during the second generation.
3. In order to test if a particular DNA sequence acts like origin of replication, we must clone
the DNA sequence in a plasmid that lacks its own origin of replication. Following
transformation in yeast, if the plasmid is able to replicate then the DNA sequence acts like
origin that directed the replication of the plasmid.
4. In case of prokaryotes, the size of Okazaki fragments varies from 1000-2000 nucleotides.
So, dividing the genome size with the size of Okazaki fragments will tell the number of
Okazaki fragments produced during replication.
5. DNA pol I plays an important role in removing the RNA primers synthesized for
production of Okazaki fragments and excicion repair So mutation in DNA pol I will result in
defects in replacing RNA by DNA and gap filling after excision repair.
30
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Mechanism of DNA replication

Subject : Molecular Biology

Lesson : Mechanism of DNA replication

Lesson Developer : Dr. Devi Lal

College / Department : Department of Zoology , Ramjas

Chapter: Mechanism of DNA replication

Topic Page No.

Raw materials for DNA synthesis………………………………………………………….

Mechanism/model for DNA replication………………………………………………………….

DNA replication follows semi-conservative model………………………………

Enzymes involved in DNA replication………………………………………………………….

DNA polymerases in prokaryotes………………………………………………………….……………………..

Family of DNA polymerases………………………………………………………….……………………………..

Sliding clamps and clamp loader………………………………………………………….………

Single stranded DNA binding protein (SSBs) …………………………………………

General principles of DNA replication…………………………………………………..

Raw material for DNA synthesis

(NMP)n + NTP (NMP)n+1 + P—P 2Pi

Mechanism/model for DNA replication

DNA replication follows semi-conservative model

E. coli grown in E. coli grown in

Parent DNA First generation Second generation

Fig. 2. The experiment of Meselson and Stahl to prove semi-conservative replication of

Watch the animation of the experiment: <iframe width="420" height="315"

Value addition: Video

Matthew Meselson (Harvard): The Semi-Conservative Replication of

Watch the talk by Meselson on Semi-conservative replication of DNA by clicking the

Enzymes involved in DNA replication

DNA polymerases in prokaryotes

S. No. Name of polymerase Family Function

Fig. 4. The structure of bacterial DNA pol III holoenzyme.

DNA polymerases in eukaryotes

Eukaryotes consist of large number of DNA polymerases as compared to prokaryotes (Table

Value addition: Interesting to know

Contributions of Arthur Korenberg

Table 2: DNA polymerases found in eukaryotes.

DNA pol α, δ and ε are involved in replication of chromosomal DNA.

S. No. Name of Family Proposed function

Family of DNA polymerases

Table 3: The family of DNA polymerases.

S. No. Class Example

6. Y Pol IV, polV, DNA pol η, DNA pol ι, DNA pol κ

Value addition: Video

Proofreading of DNA polymerase

Source: You Tube

Sliding clamps and clamp loader

As already mentioned, the processivity of DNA polymerase is increased with their

ATP ATP ATP

Clamp loader (γ-

ATP hydrolysis (D)

• DNA ligase I: links Okazaki fragments.

• DNA ligase III: involved in base excision repair.

• DNA ligase IV: involved in repair of double stranded DNA breaks

Single stranded DNA binding protein (SSBs)

Value addition: Video

General principles of DNA replication

OriC Replication fork Replication fork

Leading strand DNA

5’ Lagging strand DNA DNA polymerase III

Value addition: Interesting to know!!!!

Reiji Okazaki was born in Hiroshima in 1930. He is

Value addition: Video

leading strand and lagging strand