Chapter 2

An Insight into DNA Replication

Abstract
DNA, the genetic material, encoded for hereditary information is transferred from
parents to their offspring. For transferring of genetic material from parents to their
next generation, identical copies of DNA are made in a process named DNA
replication and are transferred from one generation to the next progeny. James
Watson and Francis Crick, after their remarkable publication on DNA structure, gave
the hypothesis of semi-conservative DNA replication. According to this hypothesis,
both strands of DNA, double stranded molecule (parent DNA strands) serve as
template for the genesis of new strands (daughter DNA strands), thereby two new
molecules of DNA are formed, so each DNA molecule possesses one new strand and
other old one. Although two other hypotheses for DNA replication; dispersive and
conservative were also proposed by other scientists yet the semi-conservative
hypothesis about DNA replication then later on was proved by Mathew Meselson
and Franklin Stahl in 1958 by performing series of elegant experiments. Hence this
chapter will provide insight into DNA replication process, both in prokaryotes and
eukaryotes in a well elaborative manner.
Keywords: DNA replication, Telomere synthesis, DNA polymerases, Telomerase,
replication competence

2.1 Introduction
DNA is the genetic material that is transferred from parents to their off-spring. Thus,
identical copies of DNA are made that are transferred to next progeny. The basics of
DNA replication are rather similar both in prokaryotes and eukaryotes though some
discrepancies are even there. In prokaryotes replication of circular DNA molecules
require an origin of replication (ori). From where two replication forks move in
opposite directions and hence they meet at a terminus region, called ter. At the end
of replication, each DNA duplex is hemimethylated at its oriC and after being fully
methylated, is ready for another round of replication. In [Link], DNA polymerase III
is the principal enzyme involved in the process of DNA polymerization and
replication. However, in eukaryotes DNA polymerase δ is principal enzyme for DNA
replication. Eukaryotic chromosomes termini have unique and special structures,
called telomeres. Telomerase enzyme synthesizes telomeres and is not dependent
upon exogenous template strand for DNA strands synthesis and uses its own RNA
molecule as a template.

11
12 Chapter 2

2.2 DNA Replication in Prokaryotes:

To illustrate the mechanism of DNA replication in prokaryotes, E. coli is presented
as model. Replication of circular DNA molecule of prokaryotes requires an origin of
replication (ori), from where two replication forks proceed in opposite directions and
result in an intermediate θ (theta) form. These replication forks, when fuse together,
mark the termination of replication process.

2.2.1 DNA polymerases in Escherichia coli

E. coli has five DNA Polymerases that are DNA-dependent DNA polymerase I,
DNA-dependent DNA polymerase II, DNA-dependent DNA polymerase III, DNA-
dependent DNA polymerase IV and DNA-dependent DNA polymerase V. These all
DNA polymerases have 3´---->5´ exonuclease (proofreading) activity because in
DNA replication a high degree of co-ordination, regulation and accuracy is required.
Therefore, for ensuring the fidelity and efficiency of DNA replication mechanism,
not only DNA polymerases as well as many enzymes, proteins and other factors
possessing activity for performing specific tasks are also involved. This entire
replication machinery forms a complex, which is termed as replisome (Griffiths et
al. 1993; Melissa and James 2007).
In [Link], DNA polymerase III is the principal enzyme involved in the process of
DNA polymerization and replication. DNA polymerase III has ten subunits i-e; α, ε,
θ, τ, γ, δ, δ́, χ, ψ and β. Subunits α and ε possess polymerization and proofreading
activities. These subunits (α and ε) along with θ subunit form core polymerase and
τ2γδδ́ subunits form clamp-loading complex. Core polymerases are linked via τ
subunit while χ subunit interacts with single strand binding proteins (SSB) and ψ
subunit interacts with γ and χ subunits (Fang et al. 1999; Kaguni 2011). This entire
assembly is DNA polymerase III with low processivity (Fig 2.1a). Processivity is
enhanced when β subunit is added and DNA polymerase III holoenzyme is formed
(Fig 2.1b).

(a) (b)
Fig. 2.1 DNA polymerase III (a) core enzyme (b) holoenzyme (Redrawn after
modification from Shevelev and Hübscher 2002).
An Insight into DNA Replication 13

Processivity is basically the ability or/ characteristic of DNA polymerases that

represent numbers of nucleotides added by polymerase enzyme before its
dissociation. Among the subunits of DNA polymerase III, β is sliding clamp that
inhibits the dissociation of this polymerase resulting in increased processivity.
During polymerization event, sometimes mistakes occur and mismatched base
pairing takes place. Hence due to the addition of wrong nucleotide, the translocation
of DNA polymerase is stalled resulting in a kinetic pause. This kinetic pause gives
the opportunity for proofreading. Thus, at this state due to the 3´‫>־־־־‬5´exonuclease
activity of DNA polymerase, a mismatched nucleotide is removed and a correct one
is inserted and polymerization process again begins (Bielinsky and Gerbi 2001;
Nelson and Cox 2012).
Another important polymerase is DNA polymerase I which is involved in clean-up
that occurs during DNA replication, recombination and DNA repair. DNA
polymerase I also has polymerization and a 3´‫>־־־־‬5´exonuclease (proofreading)
activity. In addition to these activities, this enzyme also has a striking, distinct and
important feature, not present in other DNA polymerases (DNA polymerase II, DNA
polymerase III, DNA polymerase IV, and DNA polymerase V). This imperative
characteristic of DNA polymerase I is 5´---->3´exonuclease activity that helps this
enzyme for replacing primers (either DNA or RNA) and performing nick translation
(Diffley 2011; Leman and Noguchi 2013).
DNA replication occurs mainly in three steps i-e., Initiation, Elongation and
Termination. These three stages will be discussed one by one.

2.2.2 Initiation of DNA Replication

The chromosomal origin of replication in [Link] is oriC, from where replication
process is started bidirectionally by a couple of replisome that are positioned on it.
The consensus sequence of oriC is ~245bps long containing 3 AT rich repeats of 13
base pairs (GATCTNTTNTTTT) and multiple repeats of 9 base pairs
(TTATCCACA) long sequences. DNA of oriC is methylated by Dam (DNA adenine
methylation) methylase. This origin of replication is methylated at N 6 position of
adenine of 5´ GATC 3´sequence in oriC. Duration of initiation event mainly depends
on the degree of DNA methylation at oriC. At the start of DNA replication, oriC –
proteins complex (Orisome) is formed in which oriC is bound by DnaA protein; as
a result, origin recognition complex (ORC) is established. The conversion of ORC
to pre-replication (Pre-RC) complex is taken place by the recruitment of initiator
proteins or factors like HU, Fis and IHF (Integration Host Factor). This phase in
DNA replication is referred as “Licensing” (Remus and Diffley 2009; Kaguni 2011).
[Link] DnaA protein complex: A replication initiation factor
In oriC the 9 base pairs long multiple repeats are DnaA boxes (R1, R2, R3, R4 and
R5) that are recognized by DnaA protein complex. Along with these DnaA boxes,
oriC also has τ (τ1 and τ2) and I (I1, I2 and I3) DNA motifs that are also recognized
and bound by ATP linked DnaA protein complex. This complex is a replication
initiation factor consisting of 4 to 5 DnaA protein molecules. The DnaA protein
belongs to AAA+ (ATPases associated with a variety of cellular activities) protein
14 Chapter 2

family of ATPases and for its function, ATP plays a decisive role. DnaA protein
complex is bound to DnaA boxes in two states i-e., DnaA-ATP and DnaA-ADP.
DnaA-ATP is active form and has more affinity of binding to DnaA boxes. These
DnaA boxes possess high as well as low affinity for DnaA protein complex. R1, R2
and R4 boxes are of high affinity whereas R3 and R5 along with τ and I sites are of
low affinity for DnaA protein. The transition of DnaA-ATP (active state) to Dna-
ADP (inactive state) takes place by a mechanism, known as regulatory inactivation
of DnaA (RIDA). This mechanism is catalyzed by Hda, a DnaA paralog allied to β-
clamp of DNA polymerase III enzyme. DnaA linked with ATP stimulates the melting
of DNA region contiguous to its binding sites (Nielsen and Anders 2008; Leonard
and Grimwade 2009).
DnaA protein has various domains. Domain I of DnaA plays a role in the interaction
and recruitment of DnaB-DnaC complex to origin of replication. Domain III deals
with the binding and hydrolysis of ATP. Domain IV possesses activity of DnaA box
recognition. Domain I and Domain III are also involved in the oligomerization of
DnaA. In the oligomerization of DnaA, 4 to 5 DnaA protein molecules interact and
splice to form DnaA protein complex that is coupled with ATP and histone like
protein (HU) to initiate DNA replication. DnaA protein molecules, first recognizes
and binds to high affinity R1, R2 and R4 boxes and then recognize and melt the three
repeat of 13 bps long consensus sequence rich in A═T base pairs and an open
complex is formed. This process is facilitated by ATP and HU (Histon like protein)
that is DNA binding protein and stimulates initiation event (Nelson and Cox 2012;
Lodish et al. 2012).
[Link] Pre-priming complex and unwinding of DNA molecule
After the aperture of double stranded DNA, DnaC loads two ring shaped hexamer of
DnaB onto each strand of double helical DNA and a pre-priming complex is
formed. DnaC protein is necessary for the loading of DnaB protein molecules at
unwound region. DnaB protein is basically a replicative DNA helicase present in the
form of ring shaped hexameres that unwinds the DNA strands by breaking the
hydrogen bonds present between complementary strands of DNA and forming two
replication forks (junction of unwound molecule) (Remus and Diffley 2009; Lodish
et al. 2012).
Each unit of hexameric DnaB complex (replicative helicase) is of bi-lobed in shape,
consisting of large C-terminal and small N-terminal domains. DnaC interacts with
DnaB to form DnaB-DnaC complex via forming a linkage with small N-terminal
domains of DnaB molecule. DnaC that is a helicase loader acts as negative regulator
for DnaB (helicase) and suppresses helicase and ATPase activity of DnaB. This
negative regulation is required for the loading of DnaB complex onto each DNA
strand. After the loading, DnaB-DnaC complex is dissociated that is required for the
activity of DnaB. Activity of DnaB helicase generates topological stresses or
torsional strains on DNA molecules that is relieved by DNA gyrase. DNA gyrase
catenates negative supercoiling and decatenates positive supercoiling in front of the
replication fork and retains the DNA molecule in proper topological domain (Lodish
et al. 2012; Leman and Noguchi 2013).
An Insight into DNA Replication 15

When a DNA molecule becomes single stranded by the activity of DnaB (helicases)
then single strand DNA binding (SSB) proteins are involved and bind DNA as
tetramers in co-operative manner. Binding of SSB molecules to single stranded DNA
(ssDNA) possesses cooperativity regarding the binding of one tetramer of SSB that
induces the binding of more SSB tetramers to single stranded DNA that inhibit the
stresses on ssDNA by holding it in extended configuration (Kaguni 2011; Nelson and
Cox 2012).

2.2.3 Elongation phase of DNA Replication

After the unwinding of parent DNA strands by DnaB protein (helicase) and holding
of separated DNA strands by SSB, elongation event starts by the activity of primase
enzyme (DnaG) that catalyzes the reaction of primer synthesis at replication origin.
Primase is a type of DNA dependent RNA polymerase whose activity is facilitated
by additional proteins, such as DnaB, DnaT, PriA, PriB and PriC. Unification of
primase and these additional proteins, results in the formation of a protein complex
that is termed as primosome. Primosome synthesizes primer by using parent DNA
strand as template. Primer is basically a short segment of DNA or RNA and has free
3´-OH group at its end. The free 3´ end of primer is its primer terminus. Primers are
mostly a short nucleotide sequence of RNA rather than of DNA (Griffiths et al. 1993;
Lodish et al. 2012).
After the primer synthesis, DNA polymerase III binds to it, and a nucleophilic attack
(3´-OH end is nucleophile) takes place at α phosphate of deoxynucleoside 5´
triphosphate that is to be added and a phosphodiester bond is formed between the
free 3´ end of primer and 5´ phosphate group of this incoming nucleotide. During
phosphodiester bond formation, pyrophosphates (β and γ) are released due to the
hydrolysis of one phosphoanhydride bond in deoxynucleoside 5´ triphosphate that
gives energy for polymerization and condensation of monomer in polymer chain (Fig
2.2). By the activity of pyrophosphatase, pyrophosphates (ppi) are hydrolyzed
subsequently to inorganic phosphate (pi), so more energy is released that facilitates
the formation of product and polymerization reaction. The process of nucleotides
addition proceeds continuously in the same way and new DNA strands are
synthesized (Lodish et al. 2012; Nelson and Cox 2012).
In elongation process, two types of DNA strands are synthesized because both
strands of a DNA molecule serve as template and are called leading strand and
lagging strand. The DNA strand of 3´---->5´ direction is called leading strand and
new DNA strands synthesizes continuously in 5´---->3´ direction from this strand.
While replication of 5´---->3´ DNA strand is discontinuous and is called as lagging
strand. Synthesis of new DNA strand from this is more complex and occurs as a
series of segments called Okazaki fragments (Fig 2.3).
16 Chapter 2

Fig. 2.2 Process of nucleotides condensation in polynucleotide chain.

Replication of lagging strand does not take place by one primer whereas synthesis of
each Okazaki fragment involves separate primer. After the synthesis of each DNA
segment of lagging strand, RNA primer is removed by DNA polymerase I and
replaced with DNA segment. The single stranded nick in DNA backbone is stitched
by DNA ligases and broken phosphodiester bond is sealed as covalent closure. After
this reaction, DNA polymerase III dissociates from β sliding clamp. For the synthesis
of next DNA segment of lagging strand, new primer is synthesized by primosome
and DNA polymerase III associates with new β sliding clamp and β subunit is loaded
at the new primer by clamp loading complex of DNA polymerase III, and new
Okazaki fragment is synthesized. This process keeps on going and lagging strand is
An Insight into DNA Replication 17

being replicated in a discontinuous manner (Griffiths et al. 1993; Lodish et al. 2012;
Nelson and Cox 2012).

Fig. 2.3 DNA replication process: Synthesis of new DNA strands from leading and
lagging strands of double stranded DNA molecule

2.2.4 DNA Replication Termination

In DNA replication of [Link], two replication forks keep moving in opposite
directions and they meet at a terminus region, called ter. A specialized protein known
as tus (terminus utilization substance) recognizes and binds at ter region which is
basically the binding site of it. This ter-tus complex stalls the movement of both
replication forks and results in the termination of replication process (Lodish et al.
2012; Nelson and Cox 2012).

2.2.5 Post DNA replicational modification in prokaryotes

After replication process, each DNA duplex is hemimethylated in a way that old
strand is methylated and new one is non-methylated. Sequestration of
hemimethylated oriC is mediated by SeqA protein in which hemimethylated oriC is
bound with SeqA protein and then interacts with plasma membrane and after a time
span, they are released from plasma membrane, having fully methylated oriC.
Sequestration inhibits the immediate re-firing of oriC (Nielsen and Anders 2008;
Remus and Diffley 2009).
Methylation reaction is a post replicational modification that is catalyzed by Dam
methylase. After methylation, DNA is ready for another round of replication. In
prokaryotes when DNA replication ends, the newly synthesized circular DNA
molecules are still linked and present in the form of catenanes and they are separated
by the action of Topoisomerase II enzyme. This enzyme produces a break in one
DNA molecule by breaking its phosphodiester bonds and results in the segregation
of these DNA molecules (Diffley 2011; Nelson and Cox 2012).
18 Chapter 2

2.3 DNA Replication in Eukaryotes

DNA replication is more complex and highly regulated in eukaryotic system than in
prokaryotes. Eukaryotic DNA is remarkably larger than prokaryotic DNA. Hence
process of DNA replication in eukaryotes is principally similar to prokaryotic DNA
replication but with some disparities. Eukaryotic chromosome is fired at multiple
sites or origins from where DNA replication proceeds bi-directionally. These are
autonomously replicating sequences (ARS), also known as replicators.

2.3.1 Initiation of DNA replication in eukaryotes

Initiation of DNA replication in eukaryotes starts after the binding of different
replication factors to specific sequences in replicators and a multisubunit ORC
(origin recognition complex) is established. ORC is a specialized protein complex
having multiple subunits that act as initiator protein complex that also belong to
AAA+ ATPases protein family. The main discrimination between these two initiator
proteins are that the eukaryotic ORC does not melt the double stranded DNA while
DnaA protein of E. coli unwinds the DNA strands at DUE (13 base pairs DNA
sequence within the unwinding [Link] protein functions in a highly regulated
way by the interactions of other proteins. Six Minichromosome maintenance (MCM2
- MCM7) proteins form a ring shaped heterohexameric MCM complex which is
functional analogue of DnaB protein of prokaryotes and acts as helicase (Bielinsky
and Gerbi 2001; Remus and Diffley 2009).
Although DnaB and MCM2-7 both are DNA replicative helicases, but their
mechanisms of loading to DNA strands are different. DnaB is loaded onto single
stranded DNA while MCM2-7 complex is loaded onto double stranded DNA.
Together with ORC, loading of MCM complex to double stranded DNA is mediated
by two proteins; Cdc6 and Cdt1. These proteins recognize and bind to ORC and then
load MCM helicase complex to DNA. Cdc6 and Cdt1 work like DnaC protein of
prokaryotes. After the recruitment of MCM2-7 complex on replicator, pre-replication
complex (pre-RC) is formed and replication origin or site achieves replication
competence or licensing, and is ready for firing that is mediated by a series of
phosphorylation events. These phosphorylation events, necessary for the recruitment
and activation of MCM complex as well as for the firing of replicator and these
events are regulated by CDK (Cyclin dependent kinases) activities (Bielinsky and
Gerbi 2001; Leman and Noguchi 2013).
Heterohexameric MCM complex breaks the hydrogen bonds present between
complementary strands of DNA molecule and double stranded DNA becomes single
stranded. These single stranded DNA is more prone to any environmental and
topological strains, so here a specialized protein, Replication Protein A (RPA) binds
to single stranded DNA and serves like single strand binding protein (SSB) of
prokaryotes (Lodish et al. 2012; Nelson and Cox 2012).
An Insight into DNA Replication 19

2.3.2 DNA polymerases in Eukaryotes

In eukaryotes, five DNA polymerases have been documented. These are DNA
polymerase α, DNA polymerase δ, DNA polymerase ε, DNA polymerase β and DNA
polymerase γ. DNA polymerase γ is DNA replicating enzyme of mitochondria and
DNA polymerase β possesses DNA repair activity, while other polymerases are for
nuclear DNA replication (Kornberg and Baker 1992). DNA polymerase α is believed
to be involved in primer synthesis and acts as replicative primase. The primase
subunits of DNA polymerase α are of ~ 50 KD and ~ 60 KD but its catalytic core
subunit is of ~180 KD, possessing polymerase activity (Kornberg and Baker 1992).
This polymerase has no 3´---->5´ exonuclease (proofreading) activity, possessing
low or even no fidelity for DNA replication, therefore it only involves in the synthesis
of primer.

2.3.3 Elongation phase of DNA replication in Eukaryotes

After primer synthesis, elongation of DNA strands start, and is mediated by DNA
polymerases ε and δ; principal enzymes for eukaryotic DNA replication. DNA
polymerase δ is considered to be the enzyme that executes the replication of leading
strand. This concept has been broken and is proved that DNA polymerase ε is
involved in the replication of leading strand whereas DNA polymerase δ performs
the synthesis of lagging strand by generating Okazaki fragments. DNA polymerase
δ has two domains; one is of ~ 125 KD and the other is of ~ 50 KD. During elongation
process, Proliferating Cell Nuclear Antigen (PCNA) interacts with DNA
polymerases and stimulates them by enhancing their Processivity. PCNA is a
homotrimer of ~37 KD having three dimensional structures. This is a circular sliding
clamp loaded by a clamp loader that is a protein named, replication factor C (RFC)
that works like prokaryotic clamp loading (γ) complex. PCNA is functionally and
structurally similar to β sliding clamp of bacterial DNA polymerase III (Kornberg
and Baker 1992; Leman and Noguchi 2013).

2.3.4 Termination of DNA replication in Eukaryotes

Eukaryotic DNA replication termination is different than prokaryotes because DNA
molecule is linear and there may be hitch of end replication because the 5́ end of
retrograde or lagging strand may remain unreplicated because there is no space for
primer annealing, so it could pose a problem of chromosome shortening. For
avoiding this problem, eukaryotic cells have a specialized system for end replication
of DNA.
[Link] Telomeres and Telomerase
Eukaryotic chromosomes termini have unique and special structures, called
telomeres, are G rich non-coding sequences that are tandemly repeated and provide
shelter against ends degradation of chromosomes that leads to the loss of genetic
information and other genome instability. Whereas, telomerase is a specialized
enzyme that performs the specific task of telomere synthesis (Watson et al. 2004).
20 Chapter 2

Telomerase is a type of reverse transcriptase enzyme that forming and replicating,

specialized structures called telomeres at the end of chromosomes. Telomerase is a
ribonucleoprotein, consisting of RNA component, TER and a protein TERT (>100
KD). Protein (TERT) component of telomerase possesses reverse transcriptase (RT)
homology domain as well as other conserved domains. The conserved domains other
than RT domain of TERT are positioned outside the RT domain and bind TER (RNA
component of telomerase). The interaction of protein with RNA is very essential for
the proper functioning of telomerase. In dimeric telomerases that have also been
found in different species, interaction of RNAs of both of its units is also very
important for the functioning of telomerases. A special feature of telomerase is
independency upon exogenous template strand for DNA strand synthesis. This
enzyme uses its own RNA molecule as a template. Like other polymerases,
telomerase is also a done extension from 3́ end. In human, at 3́ end of leading strand
a hexamer sequence i-e; 5́TTAGGG3́ is present. The leading strand is synthesized
continuously and is extended. Telomerase enzyme recognizes 5́TTAGGG3́ sequence
at 3́ end of leading strand because it is complementary to the RNA component of
telomerase i-e 5́CCCUAA3́. This RNA component anneals with 3́ end in such a way
that the end of RNA remains single stranded, and serves as a template for the addition
of complementary nucleotides (Fig 2.4). A small section or region of TER (RNA of
telomerase) that acts as template to synthesize or copy the telomeric DNA is called
templating domain. This synthesis of telomeres is continued hence a very
specialized system; telomere homeostasis, regulates the telomeres extension and
prevent the over extension of telomeric region. This system also encourages the
extension of telomeres when these become short (Blackburn 2005; Zvereva et al.
2010).
When this sequence is synthesized tandemly then telomerase is dissociated by
breaking DNA:RNA link, shows that this enzyme has helicase activity too because
it disrupts and breaks the hydrogen bonds present between DNA:RNA duplex and
unwinds it. After dissociation, telomerase again anneals with newly synthesized
telomere and starts the synthesis of next telomere in the same way and this process
goes on continuously and 3́ end is extended and telomere sequence is repeated
tandemly for hundreds of times. This extended 3́ end provides a room for replisome
of lagging strand and serves as template for the synthesis of 5́ end of lagging strand
and genetic information remains saved and secured from lost (Lodish et al. 2012;
Nelson and Cox 2012).
An Insight into DNA Replication 21

Fig. 2.4 Synthesis of telomeres by telomerase (Conceived from Watson et al. 2004)

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22 Chapter 2

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