Unit 11 DNA Replication

UNIT 11
DNA REPLICATION

Structure
11.1 Introduction 11.5 DNA Replication in
Eukaryotes
Objectives
Multiple Replicons
11.2 Types of DNA replication
Two or More DNA
Semi Conservative
Polymerases
Replication
Duplication of Nucleosomes at
11.3 Models of Replication
Replication Fork
Theta (Ǿ) Model of Replication
Telomerase Enzyme
Uni and Bidirectional
11.6 Summary
Replication
11.7 Terminal Questions
Rolling Circle Replication
11.8 Answers
11.4 DNA Replication in
Prokaryotes
Initiation of Replication
Unwinding of DNA
RNA Priming
Elongation of DNA Chain
Proof Reading
Termination of Replication

11.1 INTRODUCTION
In the previous units you have studied about DNA. DNA carries all of the
genetic information that is transmitted to the daughter cells after division. The
knowledge of the structure of DNA enabled scientists to work out the detailed
mechanisms of processes such as DNA replication and recombination. During
replication, DNA makes a copy of itself by copying the genetic
information. DNA replication is also referred as duplication. Before each
division, duplication of DNA molecules takes place by replication resulting in
formation of copies of molecules. DNA replication is the process by which
DNA makes a copy of itself during cell division. In prokaryotes, a single,
double-stranded DNA molecule is present in the form of a loop or circle. In 51
Block 3 DNA-Blueprint of Life
contrast there are several double-stranded linear DNA molecules present in
the genome of eukaryotes. In this unit you will be studying about the process
of DNA replication in detail.

Objectives
Objectives
After studying this unit, you would be able to:

explain DNA replication in Prokaryotes;

describe DNA replication in Eukaryotes;

enlist various enzymes involved in replication; and

describe replication of linear and double stranded (ds) DNA.

11.2 TYPES OF DNA REPLICATION

Watson and Crick proposed the double helical structure of DNA with
complementary base pairing. The structure of DNA suggested the possible
mechanism for replication of DNA molecules. Three models of DNA replication
were suggested namely conservative, dispersive and semi-conservative (Fig.
11.1). In conservative type of replication, double stranded molecule is
conserved as such and a new copy of DNA is synthesized from the old
molecule. In dispersive mode of replication, the old molecule disintegrates and
two molecules are synthesized, while in semiconservative type of replication,
the two strands separate from each other, maintain their integrity and
synthesize its complementary strand from the pool of nucleotides (Fig. 11.1).

52 Fig.11.1: Diagram showing different modes of DNA replication.

Unit 11 DNA Replication
The newly synthesized molecule would carry or conserve one of the two
strands from the parent molecule and the other would be newly synthesized.

11.2.1 Semi Conservative Replication

Watson and Crick proposed that two complementary strands of the DNA
double helix unwind and separate. Each strand guides the synthesis of a new
complimentary strand. The sequence of bases in each parental strand is used
as a template. The template guides the sequence of bases in the newly
synthesized strand. This mechanism of DNA replication is called semi
conservative replication. In this type of replication, half of the parental
molecule is conserved. In 1958, Matthew Meselson and Franklin Stahl
demonstrated that chromosome of E. coli replicate in a semiconservative
manner.

Meselson-Stahl Experiment

Meselson and Stahl proved that DNA replication was semiconservative using
nitrogen which is a major constituent of DNA. They grew E. coli in a medium
containing 15NH4Cl. The medium contained a heavy isotope of nitrogen, 15N
(14N is the naturally abundant isotope). After growing the bacteria on 15N
containing medium for several generations, they found that DNA of cells had a
high density (heavier). Meselson and Stahl transferred the bacteria grown on a
15
N medium to a medium containing only 14N. The new DNA, replicated in the
14
N medium, showed density intermediate between those cells grown
independently on light (14N) and heavy (15N) medium. Since the replication was
semiconservative, double stranded DNA was synthesized in which one strand
had 15N DNA and the other strand was having 14N DNA. If replication had been
conservative, two strands have either heavy (15N) or light (14N) DNA (Fig.11.2).
If the method of replication had been dispersive, various multiple-banded
patterns would have appeared, depending on the degree of dispersiveness.

The density of the strands was determined using a technique known as

density-gradient centrifugation. In this technique, a cesium chloride (CsCl)
solution is spun in an ultracentrifuge at high speed for several hours.
Eventually a density gradient is established in the tube with an increasing
concentration of CsCl from top to bottom. DNA formed a band in the tube at
the point where its density is the same as that of the CsCl. If DNA with
different densities are added, then several bands are formed.

Meselson and Stahl took cells growing in the medium containing 15N for
several generations, washed them to remove the medium containing 15N and
transferred them to medium containing 14N. The cells were allowed to grow in
the presence of 14N for varying periods of time. The DNA was extracted and
analyzed using CsCl density gradient. The result indicated semi conservative
mode of DNA replication. The DNA isolated from cells had a density half way
between the densities of heavy and light DNA. The intermediate density is
called as hybrid density. After two generations of growth in the medium
containing 14N, half of the DNA showed hybrid density (15N-14N) and the other
half showed light density (14N). The results proved the semiconservative
replication in DNA. 53
Block 3 DNA-Blueprint of Life

Fig. 11.2: Meselson-Stahl experiment.

DNA replication in eukaryotes is also semi conservative. This was

demonstrated at the chromosome level through experiments carried out by J.
Herbert Taylor, Philip Woods and Walter Hughes in 1957. They took root tip
cells of Vicia faba. They grew the root tips for eight hours in a medium
containing radioactive 3H- thymidine. The root tips were washed and
transferred to a non-radioactive medium containing alkaloid colchicines.
Colchicine binds to microtubules and prevents the formation of functional
spindle fibers. Therefore, the daughter chromosomes do not undergo the
separation during anaphase. The numbers of chromosomes double per cell
cycle. The doubling of chromosomes number per cell generation helped in
determining number of times DNA duplication each cell has undergone
subsequent to incorporation of radioactive thymidine. Taylor and his
colleagues used technique of autoradiography to examine the radioactivity in
the chromosomes of cells of the first metaphase, second metaphase and so
on. In autoradiography technique, the radioactive isotopes are used in the
cytological preparations of macromolecules. The radioactive atoms are
exposed to photographic film. The radioisotopes are detected by exposing
them to photographic emulsion that is sensitive to a low energy radiation. The
emulsion contains silver halides that produce tiny black spots called silver
grains when exposed to the charged particles emitted during the decay of
radioisotopes. The silver grains visible on the film are counted and each grain
54 of silver represents a radioactive decay. The radioactive decay provides an
Unit 11 DNA Replication
estimate of the quantity of radioactive material present. The technique of
autoradiography proved useful in studying DNA metabolism as DNA are
specifically labelled by 3H- thymidine. Thymidine is incorporated into DNA.
Taylor and his colleagues concluded that chromosomal DNA in V. faba
segregated in a semi conservative manner during each cell division.

11.3 MODELS OF REPLICATION

Various models have been proposed to explain the semi-conservative
replication of DNA in viruses, bacteria and eukaryotes. You will not study
these models in detail.

11.3.1 Theta (Ø) Model of Replication

In 1963, J. Cairns verified the semi conservative method of replication
photographically using autoradiography technique. Cairns grew E. coli bacteria
in a medium containing radioactive thymine, a component of one of the DNA
nucleotides. As the DNA replicated, radio labelled tritium (3H)/ tritiated
thymidine got incorporated into the newly synthesized DNA at the place of
thymine which is present opposite to adenine. Cairns extracted DNA from
bacteria and placed it on photographic emulsion for a period of time. He
examined the autoradiographs under the electron microscope. The study of
the autoradiograph revealed that DNA of E. coli is circular and replicates
maintaining the integrity of the circle (Fig. 11.3). The circular DNA does not
break during the process of DNA replication but an intermediate theta
structure is formed (similar in shape to the Greek letter theta).

Fig. 11.3: Autoradiograph developed during Cairns experiment.

The replication initiates from the unique origin of replication (ori C) site, which
is preceded by the formation of a Y-shaped structure called the replication
fork of the DNA. The DNA is unwound at origin site, and replication proceeds
in a semiconservative manner in the direction of the replication fork. Each ori
C site shows two Y-shaped replication forks clearly indicating the bidirectional
replication of DNA (Fig. 11.4). 55
Block 3 DNA-Blueprint of Life

Fig. 11.4: Theta model of DNA replication.

Every newly synthesized DNA has one parental template strand and one
daughter strand. The replication always initiates from a specific point in DNA.
John Cairns established the existence of a site of initiation or origin of
replication on the circular chromosome of E. coli. There is one unique origin
per chromosome which controls the replication of whole chromosome. The
single origin of replication, called ori C, in E. coli has been characterized. It
has been found to be 245 nucleotide pairs long with two different conserved A:
T rich repeat sequences. The region where the double helical structure opens
for replication is called replication bubble/eye. In prokaryotes one bubble is
seen. Each replication bubble has two replication forks moving and
synthesizing DNA in two opposite directions. The replication of DNA in E. coli
occurs at the time of cell division.

11.3.2 Uni and Bidirectional Replication

The replication in DNA can be unidirectional or bidirectional. This depends
upon whether the replication from the point of origin proceeds only in one
direction or proceeds in both the directions. Replication eye may appear in
both the types unless the replication starts from one of the two ends of a linear
56
DNA molecule. In unidirectional replication, one of the two ends of the
Unit 11 DNA Replication
replication eye will be stationary and the other end will move with replication.
On the other hand, in bidirectional replication, both ends will be moving and
none will be stationary (Fig. 11.5). Distinction between unidirectional and
bidirectional replication can be made by the study of autoradiographs If
radioactively labelled nucleotides are used during DNA synthesis.

Fig. 11.5: a) Diagrammatic representation of the uni and bidirectional DNA

replication; b) bidirectional DNA replication

The structure of bacterial chromosome was determined by John Cairns in

1963 with the help of autoradiography technique. He grew E. coli cells in
medium containing 3H –thymidine for varying period of time. He carefully
collected chromosomes on membrane filters. These filters were stored in dark
for a period of time to allow sufficient radioactive decay. The autoradiographs
showed that chromosomes of E. coli are circular structures that exist as theta
(θ) shaped intermediates during replication. The auto radiographs indicate that
unwinding of two complementary strands of DNA and their semi conservative
replication occur simultaneously or are closely coupled. The parent double
helix rotates about 360o to unwind each strand of helix to produce some kind
of swivel (turn around a point or axis). The enzyme topoisomerases is
responsible for producing transient breaks in the DNA that acts as a swivel for
DNA unwinding and keep the DNA untangled. Enzyme topoisomerases
provide the required axes of rotation during replication of circular DNA 57
Block 3 DNA-Blueprint of Life
molecules. The enzyme catalyzes the transient break in DNA molecules which
provides an axis of rotation that allows segments of DNA on opposite sides to
break. The replication of circular chromosome in E. coli is bidirectional starting
from the unique origin of replication. Two replication forks move in opposite
directions sequentially around the circular chromosome. Hence Cairns
experiment suggested that replication of E. coli chromosome advances in both
directions away from the origin of replication.

Box 11.1 : Topoisomerase

Enzyme Topoisomerases change the state of supercoiling in a DNA molecule.

Topoisomerases were discovered by Martin Gellert and James Wang. They are of
two types - type I or type II. Topoisomerase I make a nick in only one strand of the
DNA molecule through which passes the intact strand whereas Topoisomerase II
cuts both the strands of DNA creating a passage through which the loop of the other
segment of helix passes. In E. coli, a Type II Topoisomerase called DNA gyrase is
found (Fig. 11.6). The enzyme relieves the mechanical strain that builds up during
replication in E. coli. The enzyme travels along the DNA ahead of the replication fork,
removing the supercoils. DNA gyrase does this by cleaving both strands of the DNA
duplex, passing a segment of DNA though the double stranded break to the other
side, then sealing the cuts. The process is driven by energy released from hydrolysis
of ATP. The movement of replication fork generates positive supercoils in the
unreplicated portion of DNA ahead of the fork.

Fig. 11.6: Mechanism of topoisomerase action.

11.3.3 Rolling Circle Replication

Rolling circle type of replication is noted in many viruses. This type of
replication is used by viruses to duplicate their genomes and bacteria to
transfer DNA from donor cells to recipient cells during genetic exchange. It has
also been noted in amphibians that amplify extrachromosomal DNAs carrying
clusters of ribosomal RNA genes during oogenesis. Rolling- circle replication
is a mechanism for replicating circular DNA molecules. The unique aspect of
this replication is that one parental circular DNA strand remains intact and rolls
or spins while serving as the template for the synthesis of a new
complementary strand. The first step in rolling circle replication is the
generation of a nick by sequence-specific endonuclease in one strand at the
origin, producing 3΄-OH and 5΄-phosphate termini. The 5΄ terminus is
displaced from the circle as the intact template strand rolls about its axis.
Covalent extension occurs at the 3΄-OH of the cleaved strand. Since the
circular template DNA may turn 360° many times, with the synthesis of one
complete or unit length DNA strand during each turn, rolling circle replication
generates single- stranded tails longer than the contour length of the circular
58 chromosome. This type of replication can produce either single- stranded or
Unit 11 DNA Replication
double- stranded progeny DNAs. Circular single – stranded progeny
molecules are produced by site-specific cleavage of the single- stranded tails.
This is followed by recircularization of the resulting molecule at the origin of
replication (Fig. 11.7). The enzymes involved in the process are the same as
those involved in θ- model of replication.

Fig.11.7: Rolling circle replication of DNA.

Box 11.2 : Endonuclease

Endonuclease is a restriction enzyme produced by bacteria that cleaves double-

stranded DNA at specific sites along the molecule. When a restriction endonuclease
recognizes a sequence, it snips through the DNA molecule by catalyzing the
hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond
between adjacent nucleotides.

SAQ 1
a) Differentiate between:

i) conservative, semi conservative and dispersive type of DNA

replication

ii) unidirectional and bidirectional replication

b) Write the functions of the following:

i) Replicon

ii) Endonuclease

iii) Topoisomerase

11.4 DNA REPLICATION IN PROKARYOTES

In prokaryotes such as E. coli DNA replication occurs in a semi conservative
manner. This refers to a replication process in which two strands separate
from one another, maintain their integrity and each strand synthesizes a new
complementary strand from the pool of nucleotides. Studies have shown that
bacteria undergo bidirectional, semi conservative mode of replication. 59
Block 3 DNA-Blueprint of Life
11.4.1 Initiation of Replication
The replication of DNA begins with unwinding of the double helix at sites
called origin of replication. At these sites, the hydrogen bonds between the
bases are broken and paired bases separate. The sites where the pair of
replicated segments come together and join the non-replicated DNA are called
as replication fork. In bacterial chromosome, DNA replication always begins
at specific sites called the origin. Each origin controls the replication of a unit
of DNA called a replicon. The bacteria have a single specific origin of
replication (Fig. 11.8). The origin of replication in E. coli chromosome has
been recognized as a specific sequence called ori C. The ori C is a 9 base
pair (bp) sequence that is repeated four times within the region. At this site the
proteins bind and initiate the process of replication. The replication fork is
formed at this site. Replication fork is a site where the parental double helix
undergoes strand separation and nucleotides are incorporated into newly
synthesized complimentary strands. The replication proceeds outward from
the origin in both the directions (bidirectional). Two replication forks move in
opposite directions until they meet at a point across the circle from the origin
where the replication is terminated. The newly synthesized strands detach
form each other and move to the different cells.

Fig. 11.8: Diagram showing origin of replication of DNA in prokaryotes at site

ori C.

11.4.2 Unwinding of DNA

For replication, the strands from the DNA duplex need to be separated. The
separation of the strands of a double helix is called unwinding. The separation
of strands at one end is accompanied by rotation of entire fiber as it resists
development of tension. The unwinding of DNA strands is accomplished by an
enzyme DNA helicase. The enzyme DNA helicases bind to the double
stranded DNA and help in separation of two strands (Box 11.3). Energy
released by ATP hydrolysis is used in the process of unwinding. The hydrogen
bonds that hold the two strands together are broken exposing the two
separated single strands as DNA templates. E. coli has 12 different helicases
60 that take part in various aspects of DNA metabolism. In addition to helicase,
Unit 11 DNA Replication
unwinding the duplex and separation of strands require the assistance of a
type of proteins called single stranded DNA binding proteins (SSB). The
unwound section appears as a bubble and is known as replication bubble.
As the strands unwind, enzyme DNA gyrase catalyses the formation of
negative supercoils which helps in the process of unwinding of DNA. The
enzyme prevents accumulation of twists by making temporary single strand
nicks in the DNA.

Box 11.3: Role of Dna B helicase in DNA replication

One of the helicases that is the product of dnaB gene helps in unwinding of DNA
during replication. The enzyme Dna B helicase consist of six subunits that form a ring
shaped protein that encircle single DNA strand. Replication in E. coli begins when
the multiple copies of DnaB (a 52KD protein) bind to the AT rich origin of replication
i.e. ori C site and causes 45bp of DNA to separate into a single DNA strand. The
DnaB helicase is then loaded on to the single stranded DNA of the lagging strand of
ori C with the help of protein DnaC. The DnaB helicase then translocates in a 5ˊ→ 3ˊ
direction along with the lagging strand template, unwinding the helix as it proceeds.
Unwinding of DNA by the helicase is aided by the attachment of single strand binding
proteins (SSB protein). These proteins bind selectively to a single stranded DNA
and the binding of these proteins keeps single stranded DNA in an extended state,
preventing it from becoming rewound or damaged.

11.4.3 RNA Priming

As the two strands separate or unzip, the bases are exposed, and the
enzyme DNA polymerase II moves at the point where synthesis will begin. The
short segment of RNA that provides the necessary 3ˊ OH terminus for the
initiation of DNA polymerase activity is called a primer. The primer is laid
down complementary to the DNA template by an enzyme RNA polymerase or
Primase. Hence we can say that synthesis of leading strand begins at the
origin of replication is initiated by a primase (RNA polymerase). The DNA
replication requires a template DNA strand to copy and a primer strand to
which nucleotides can be added. The enzyme DNA polymerase synthesizes
DNA in a 5ˊ→3ˊ direction. The short RNAs synthesized by the primase at the
5ˊ end of the leading strand and the 5ˊ end of the each Okazaki fragment
serve as the primer for the synthesis of DNA by DNA polymerase.

In bacteria, primase and helicase associate to form a protein complex called

primosome. Primosome is involved in the synthesis of RNA primer sequences
used in DNA replication. It moves along a DNA molecule with the help of
energy of ATP. The helicase moves along the lagging strand template, while
primase binds to helicase periodically and synthesizes short RNA primers that
begin the formation of Okazaki fragments. The discovery that DNA gets
synthesized as short fragments due to discontinuous replication was made by
Reiji Okazaki. The initiation of Okazaki fragments on the lagging strand is
carried out by primosome (Fig.11.9). As it proceeds, the DNA helicase
unwinds the parental DNA double helix, and synthesizes RNA primers needed
for the discontinuous synthesis of the lagging strand. DNA primase
synthesizes RNA primers that are covalently extended with the addition of
deoxyribonucleotides by DNA polymerase III. Single stranded DNA binding
protein coats the unwound unreplicative DNA and keeps it in an extended
state for DNA polymerase III. The RNA primers are replaced with DNA by 61
Block 3 DNA-Blueprint of Life
DNA polymerase I and the single strand nicks left by polymerase I are sealed
by DNA ligase. The complete replication apparatus moving along the Dnb A
molecule at replication fork is called replisome. The replisome contains DNA
polymerase III holoenzyme, two catalytic cores one replicating the leading
strand and the other replicating the lagging strand.

Fig. 11.9: Diagram showing priming during DNA replication in prokaryotes.

11.4.4 Elongation of DNA Chain

DNA Polymerase III is the enzyme mainly involved in DNA replication. It
performs 5ˊ→3ˊ polymerase function. It is also known as replicative
polymerase. Specific activity of DNA polymerase III helps in extending the
RNA primers. DNA polymerase III starts adding nucleotides one by one in a
complementary manner i.e. A to T and G to C. The action of DNA polymerase
is dependent on template. It reads the sequence of bases on the template
strand and synthesizes the complementary strand. The template strand is
always read in the 3ˊ→5ˊ direction. The new DNA strand is synthesized in the
5ˊ→3ˊ direction. DNA polymerases III also catalyzes the formation of hydrogen
bonds between the new nucleotides and the nucleotides on the template
strand. The enzyme also catalyzes the reaction between 5ˊ phosphate on a
new nucleotide and free 3ˊ OH on the growing polynucleotide (phosphodiester
bond), therefore the new DNA strands grow in 5ˊ→3ˊ direction. The growth of
the strand begins at 3ˊ OH group of the sugar and 5ˊ phosphate group of the
newly synthesized nucleotide. DNA polymerase III helps in the synthesis of
successive fragments for the lagging strand.

DNA strands are complementary and run antiparallel to each other. Only one
new strand at the 3ˊ end of the template DNA grows continuously in the
opposite direction because it is complementary to the template strand. The
strand that is synthesized continuously is called the leading strand and the
other strand, synthesized discontinuously in the form of small fragments, is
62 called the lagging strand (Fig. 11.10). As a result of discontinuous replication,
Unit 11 DNA Replication
short Okazaki fragments are synthesized. Okazaki fragments usually have
1000 to 2000 nucleotides in prokaryotes while in eukaryotes they are smaller
in size ranging from 100 to 200 nucleotides. DNA polymerase III molecule
synthesizes successive fragments of the lagging strand. The polymerase III
molecule is recycled from the site where it has just synthesized one Okazaki
fragment to the next site along the lagging strand template closer to the
replication fork. At the new site, the polymerase attaches to the 3΄-OH group of
the RNA primer laid down by a primase.

Fig. 11.10: Diagrammatic representation of the replication fork formed during

DNA replication.

The primers associated with Okazaki fragments at its 5΄end are removed and
the resulting gaps are filled with DNA with the help of DNA polymerase I (Fig.
11.11). The ends of each fragment are later joined by DNA ligase to form a
continuous strand (lagging strand). The enzyme joins the fragments together
by forming the phosphodiester bonds. DNA ligase helps in sealing the gaps or
nicks when RNA primer is removed.

Two polymerases move in opposite directions with respect to the linear axis of
the DNA molecule. Once the DNA polymerase III, synthesizing an Okazaki 63
Block 3 DNA-Blueprint of Life
fragment on the lagging strand template, approaches the 5΄ end of the
previously synthesized Okazaki fragment, the lagging strand template is
released and the polymerase begins work at the 3΄ end of next RNA primer
towards the replication fork (Fig.11.11).

Fig. 11.11: Mechanism of DNA replication in prokaryotes.

Box 11.4: DNA polymerase I

DNA polymerase I was the first enzyme with the polymerase activity. It was isolated
by Arthur Kornberg in 1960. The enzyme plays a role in 5ˊ→3ˊ elongation
(polymerase activity), 3ˊ→5ˊ exonuclease (proof reading activity) and 5ˊ→3ˊ
exonuclease activity (DNA repair). These activities are confined to three active sites
present on the large fragment of enzyme called Klenow fragment. DNA polymerases
need to remain associated with the template to synthesize a continuous
complementary strand and must get attached to the template to move from one
nucleotide to the next. DNA polymerase I shows both 5ˊ→3ˊ and 3ˊ→5ˊ exonuclease
activity. Being exonuclease DNA polymerase is able to degrade DNA polymers by
removing one or more nucleotides from the end of the molecule.

Semidiscontinous replication

The newly synthesized strand is synthesized only in 5ˊ→3ˊ direction. One

strand i.e. the leading strand is synthesized continuously because its synthesis
continues as the fork advances, while the other strand i.e. the lagging strand is
synthesized discontinuously because the initiation of each fragment waits for
the parental strand to separate and expose the additional template
(Fig. 11.12).

Fig. 11.12: Diagrammatic representation of Continuous and discontinuous type

64 of DNA replication.
Unit 11 DNA Replication
Since one strand is synthesized continuously and other discontinuously, the
replication is called as semidiscontinuous. The leadings strand shows
continuous replication by adding nucleotides to the growing end while the
lagging strand shows discontinuous replication by adding short nucleotides
segments to the growing end (Fig.11.13).

Fig. 11.13: Comparison of different types of DNA replication.

11.4.5 Proof Reading

During DNA duplication, one error occurs in every billion base pairs. This
fidelity is necessary to minimize the occurrence of mutations, especially in
large genomes. The problem of DNA replication is solved by a mechanism
called proofreading. The process of proof reading involves scanning of termini
of nascent DNA chains for errors and correcting them. The process is carried
out by 3ˊ→5ˊ exonuclease activity of DNA polymerase. This helps them to
detect and excise a mismatched nucleotide in the 3΄→5΄ direction. When a
template primer DNA has a terminal mismatch (an unpaired or incorrectly
paired base or sequence of bases at 3ˊ end of primer), the 3ˊ→5ˊ exonuclease
activity of DNA polymerase clips off the unpaired base or bases. Once the
mismatched nucleotide is removed, DNA synthesis proceeds in 5΄→3΄
direction. In E. coli, the proof reading function is carried out by ε subunit of
DNA polymerase II.

Hence, high fidelity during DNA replication is based on the fact that DNA
polymerase does not simply catalyze the incorporation of nucleotide at the
right place, it also actively discriminates against incorporation of a mismatched
base by adapting to the conformation of a correct base pair.

11.4.6 Termination of Replication

The meeting of DNA replication forks results in termination of DNA replication
(Fig. 11.14). The formation of the replication fork stops when a protein called
replication terminator protein binds to specific sites on a DNA molecule. It 65
Block 3 DNA-Blueprint of Life
has been found that proteins, ter A and ter B block the movement of
replication forks advancing in the clockwise and anticlockwise direction at
variable sites. Before completion of DNA replication, the enzymes are used to
proof read the sequences to make sure the nucleotides are paired up correctly
in a process called DNA repair. If any mistake occurs during the DNA
replication, the enzyme nuclease removes the incorrect DNA. DNA
polymerase II plays a role in DNA repair and filling up the gaps.

Fig.11.14: Diagrammatic representation of DNA replication in bacteria.

A series of enzymes are required in unwinding and separation of double

stranded DNA molecule (Table 11.1).

Table 11.1: Role of different enzymes used in DNA replication.

Enzyme
Function in DNA Replication

DNA Helicase Also known as helix destabilizing enzyme. Unwinds the DNA
double helix at the Replication Fork.

DNA Polymerase Synthesizes new duplex DNA strand by adding nucleotides in

the 5ˊ to 3ˊ direction. Also performs proof-reading and error
correction.

Primase Provides a starting point of RNA (or DNA) for DNA polymerase
to begin synthesis of the new DNA strand.

DNA Gyrase A kind of topoisomerase that removes DNA supercoils ahead

of replication fork.

Topoisomerase Relaxes the DNA from its super-coiled nature.

Single-Strand Bind to ssDNA and prevent the DNA double helix from re-
Binding (SSB) annealing after DNA helicase unwinds it, thus maintaining the
Proteins strand separation, and facilitating the synthesis of the nascent
strand.

DNA clamp A protein which prevents DNA polymerases from dissociating

from the DNA parent strand during the process of replication.

DNA Ligase Seals the gaps between the Okazaki fragments to create one
continuous DNA strand

Telomerase Lengthens telomeric DNA by adding repetitive nucleotide

sequences to the ends of eukaryotic chromosomes.
66
Unit 11 DNA Replication
Various steps involved in DNA replication in prokaryotes can be
summarized as follows:
1. The double helix structure of the DNA molecule is unzipped. This
process is carried out with the help of an enzyme helicase that breaks
the hydrogen bonding between the complementary bases.
2. The separation of DNA strands into two single strands creates a ‘Y’
shaped replication ‘fork’. Two single strands act as templates for making
the new strands of DNA.
3. One of the strands is oriented in the 3ˊ→5ˊ direction (towards the
replication fork). This is referred as the leading strand. The other strand
is oriented in the 5ˊ→3ˊ direction (away from the replication fork). This is
called as lagging strand. Because of the difference in orientation, the two
strands replicate differently.
4. A fragment of RNA called Primer comes along and binds to the leading
strand. The primer starts the DNA synthesis. DNA polymerase binds to
the leading strand and moves ahead adding new complementary
nucleotide bases to the DNA strand in the 5ˊ to 3ˊ direction (Fig. 11.15).
This type of replication is called continuous.
5. Numerous RNA primers (synthesized by enzyme primase) bind at
various points to the lagging strand. Fragments of DNA, called Okazaki
fragments, are then added to the lagging strand also in the 5ˊ→3ˊ
direction. This type of replication is called discontinuous as the
fragments will need to be joined up later (Fig. 11.15).
6. The bases are matched up (A with T, C with G) with the help of enzyme
called exonuclease that removes the primer(s). The gaps are filled by
complementary nucleotides. The new strand is proofread to make sure
there are no mistakes in the new DNA sequence.
7. Enzyme called DNA ligase seals up the sequence of DNA into two
continuous double strands. Hence after DNA replication, DNA molecule
formed consists of one new and one old chain of nucleotides. Hence the
DNA replication is described as semi-conservative.

Fig. 11.15: An overview of DNA replication in prokaryotes. 67

Block 3 DNA-Blueprint of Life

SAQ 2
a) Fill in the blanks:

i) The site of origin of replication in prokaryotes such as E. coli has

been recognized as…………………… .

ii) The process of separation of two strands of DNA is referred as

……………….. .

iii) ……………… enzyme helps in the unwinding of strands of a

double helix DNA.

iv) Enzyme DNA gyrase induces formation of ……………… that

helps in unwinding of DNA.

v) The short segment of RNA that provides the necessary 3ˊ OH

terminus for the initiation of DNA replication is called …………...

vi) Primase provides a …………….. segment for DNA polymerase to

begin synthesis of new strand.

vii) …………………. enzyme catalyzes the formation of hydrogen

bonds between the new nucleotides and helps in elongation of
DNA strand.

viii) The process that involves scanning of termini of nascent DNA

chains for errors and correcting them is called as ………………….

b) Explain the following terms:

i) Primosome

ii) Okazaki fragments

iii) Exonuclease activity

The first experimental

evidence for 11.5 DNA REPLICATION IN EUKARYOTES
bidirectional
replication in The DNA replication in eukaryotes occurs in a semi conservative manner. In a
eukaryotic cells was semi conservative mode, two complementary polynucleotide chains are
obtained by synthesized on a template strand. The parental DNA strand undergoes
autoradiography of cleavage and the two new double helices formed that carry old fragments and
labelled DNA
the newly synthesized DNA (Fig.11.16). Since the parental strands are
molecules obtained
from cultured
dispersed into two newly synthesized helices it is called dispersive replication.
mammalian cells. The
Though most aspects of DNA replication are similar in prokaryotes and
studies revealed
eukaryotes but still some prominent differences are noted between them.
presence of clusters
of active replicons Some of the major differences have been listed below:
each having two
• The eukaryotic cells have larger genomes and complex chromosomal
growing forks moving
away from a central structures.
origin.
• DNA synthesis takes place in a small portion of the cell cycle in
68 eukaryotes. Replication occurs in the S phase of the cell cycle.
Unit 11 DNA Replication

• DNA synthesis is not continuous as in prokaryotes.

• The large DNA molecules in eukaryotes take more time to replicate of

each chromosome contained a single origin.

• The eukaryotic chromosomes contain multiple origins of replication. DNA

replication is initiated at multiple origins in a coordinated manner.

• Two or more polymerases are used for replication of leading and lagging
strands at replication fork.

Box 11.5: Autonomously replicating sequence (ARS)

The origin of replication sequences (ORIs) found in budding yeast is defined as

autonomously replicating sequences (ARSs). These sequences initiate the process
of replication. ARSs help in maintaining the stability of chromosomes and plasmids
during genome replication. In yeast ARSs were identified by while maintaining the
stable replication ability of plasmids. Replication timing and efficiency are the two
key features of ARSs. The activation timing of ARS are controlled by chromatin
environment and epigenetic modifications. ARS have been identified using DNA
microarray technology and bioinformatics. The structural and functional properties
of ARSs on yeast chromosomes are being explored. Researchers are also
interested in knowing the effects of ARSs on gene silencing and expression of
genes.

11.5.1 Multiple Replicons

In eukaryotes, very large sized chromosomes having multiple origins
collectively control the replication. Replication origin sites in eukaryotes also
have unique A:T rich sequences which facilitate the opening of double helix. In
eukaryotic chromosomes several bubbles or eyes exist. Eukaryotes show
many replicons. The multiple origins collectively control the replication of DNA
molecule.

When the two replication forks move bidirectionally from a central origin, the
DNA replication generally gets completed in 8.5 days as noted in Drosophila
melanogaster. Faster replication is achieved by initiating the DNA synthesis at
many origins of replication simultaneously. For faster replication of DNA,
largest chromosome initiates replication at multiple sites (Fig.11.16). The first
evidence of multiple origins in eukaryotic chromosomes came from the pulse
labeling experiments with Chinese hamster cells. Joel Huberman and Arthur
Riggs pulse labelled cells with 3H- thymidine for a few minutes, extracted DNA
and performed autoradiographic analysis of the labelled DNA. They observed
tandem array of silver grains indicating that DNA had multiple origins of
replication. The pulse labeling period was followed by a short interval of
growth in non radioactive medium, the tandem arrays contained central
regions of high grain density at both ends. The results indicate that replication
is bidirectional. The tails of decreasing grain density result from the gradual
dilution of the intracellular pools of 3H- thymidine by 3H- thymidine as
replication fork move bidirectionally from central origin toward replication
termini. A segment of DNA whose replication is under the control of one origin
and two termini is called replicon. 69
Block 3 DNA-Blueprint of Life

Fig.11.16: Diagrammatic representation of various origin of replication in

eukaryotes.

11.5.2 Two or More DNA Polymerases

In prokaryotes five different forms of DNA polymerases are found viz. I, II, III,
IV, V whereas in eukaryotes 15 different forms of DNA polymerases are found.
These include α, β, γ, δ, ε, ζ, η, θ, ι, κ, λ, µ, σ, REVI, TD.

The unwinding of DNA strands requires DNA topoisomerase and DNA

helicase as in prokaryotes. The unwound strands are kept in an extended
state by a single strand DNA binding protein called replication protein A. The
replication of chromosomes requires the presence of three DNA polymerase-
polymerase α (Pol α), polymerase δ (Pol δ) and polymerase ε (Pol ε). All
polymerases are present in each replication fork and each polymerase
contains multiple subunits. The replisome in E. coli contains 13 proteins while
those of yeasts and mammals contain 27 different polypeptides.

In eukaryotes, Pol α is required for the initiation of replication at origin and for
priming of Okazaki fragments during discontinuous synthesis of the lagging
strand. Pol α exists in a stable complex with DNA primase. The enzyme DNA
primase synthesizes RNA primers which are extended by
deoxyribonucleotides by Pol α to produce 30 nucleotides chain of RNA-DNA.
These RNA-DNA primers are extended by Pol δ. This enzyme completes the
replication of the lagging strand while Pol ε catalyzes the replication of the
leading strand. Both polymerases δ and ε contain 3ˊ→5ˊ exonuclease activity
required for proof reading. These enzymes cannot remove RNA primers like
DNA polymerase I found in E. coli. RNA primers are excised by nucleases,
ribonuclease H1 (which degrades RNA present in the RNA-DNA duplexes)
and ribonuclease FEN-1 (F1 nuclease I). Polymerase δ fills in the gaps and
70 DNA ligase seals the nicks.
Unit 11 DNA Replication

In prokaryotes, Polymerase I, II, III play a major role in DNA replication while in
eukaryotes it is polymerase α, β, γ, δ, ε which play a crucial role in DNA
replication and repair (Table 11.2).

Table 11.2: Role of different DNA polymerases found in eukaryotes

DNA polymerases Subunits 3ˊ to 5ˊ Function

exonuclease
activity
Alpha α 4 No RNA/DNA primers, initiation
of DNA synthesis
Delta δ 4 Yes Lagging strand synthesis,
DNA repair, proof reading
Epsilon ε 4 Yes Leading strand synthesis,
proof reading
Gamma γ 2 Yes Mitochondrial DNA
replication and repair/ editing
Beta β 1 No Base-excision DNA repair
Eta η, zeta ζ, 1,2,1,1 No Damaged/distorted
kappa κ, iota ι (translesion; TLS) DNA
synthesis
Theta θ, lambda λ, 1,1,1 No DNA repair
mu µ
Nu υ 1 No Unknown
REV1 1 No DNA repair

Box 11.6: DNA polymerase III

The active form of DNA polymerase III is known as holoenzyme. It is made up of ten
unique polypeptide subunits. The largest subunit, α, along with subunits ε and θ, form
a complex called the core enzyme, which imparts the catalytic function to the
holoenzyme. Each enzyme contains two or three core enzyme complexes. Five
subunits (γ, δ, δ΄, χ and υ) are complexed together to form slide clamp loader. This
pairs with the core enzyme and facilitates the function of a critical component of
holoenzyme called the β clamp or sliding DNA clamp. Activity of clamp loader is
driven by the energy of ATP hydrolysis. The DNA clamp resembles a doughnut
which can open, close and encircle the unreplicated DNA helix (Fig.11.17). It keeps
the DNA polymerase associated with the template DNA during polymerization of
nucleotides.

Fig.11.17: Structure of DNA Polymerase III holoenzyme.

71
Block 3 DNA-Blueprint of Life
11.5.3 Duplication of Nucleosomes at Replication Fork
As we know that DNA in eukaryotic chromosomes is packaged into bead like
structures called nucleosomes, each of which contain 166 nucleotide pairs of
DNA wound around an octamer of histone molecules. The nucleosome need
to be disassembled so that the duplication of DNA packaged in a replisome
takes place. A number of proteins play a role in the assembly and
disassembly of nucleosomes during chromosome replication in eukaryotes.
Two important ones are nucleosome assembly protein-I (Nap 1) and
chromatin assembly factors (CAF-1). Nap-1 transports histones from their site
of synthesis in the cytoplasm to the nucleus and CAF-1 carries them to the
chromosomal sites of nucleosome assembly. CAF-1 delivers histones to the
site of DNA replication by binding to proliferating cell nuclear antigen (PCNA)
the clamp that tethers DNA polymerase δ to the DNA template.

11.5.4 Telomerase Enzyme

At the end of DNA replication, RNA primers are removed with the help of DNA
polymerase I. When the primers are located at the end of the strand (telomeric
ends) they get removed and single strand overhangs are formed. DNA
polymerases cannot replicate the terminal DNA segment of lagging strand of a
linear chromosome. As at the end of DNA molecule, no free 3ˊ-OH (Primer) is
available for polymerization of doxyribonucleotides. Because of this, in the
next round of chromosome replication, shortened DNA strand serves as the
template for the synthesis of new partner strand. The new DNA duplex lacks
will lack the sequences corresponding to those of RNA primer from the
previous round of replication. This result in loss of sequences and after
successive rounds of replication, the chromosome shrinks from its ends.

The special structure of telomeres provides a mechanism for an RNA

containing enzyme called telomerase which helps to prevent the shortening of
chromosome ends. The enzyme telomerase was discovered by Elizabeth
Blackburn and Carol Greider in 1985. In human chomosomes, telomeres
contain tandemly repeat sequence TTAGGG. Telomerases recognizes G –rich
telomere sequence on the 3ˊ overhang and extends it 5ˊ→ 3ˊ one repeat unit
at a time. Telomerase does not fill in the gap opposite the 3ˊ end of the
template strand but simply extends the 3ˊ end of the template strand. The
unique feature of telomerase is that it contains an inbuilt RNA template. The
telomerases contain an integral RNA component which has a sequence
complementary to the repeat sequence that makes it act as a template for a
reverse transcriptase reaction (Fig. 11.18). The telomerase then moves to the
new 3ˊ end once the repeat has been added and does it all again. Once
several telomere repeat units are added by telomerase, DNA polymerases
catalyze the synthesis of the complementary strand. Without telomerase, the
linear chromosomes would become progressively shorter. If telomeres are not
taken care off then with each round of cell division the DNA would become
shorter. This is known as the “end-replication” problem. Abnormalities in
telomere replication and malfunctioning of telomerase have been shown to
result in several autosomal diseases resulting in premature ageing, cancers,
72 bone marrow abnormalities, oesteoporesis, and many more.
Unit 11 DNA Replication

Fig 11.18: Diagrammatic representation of mechanism of telomerase action

during DNA replication.
The process of DNA replication in eukaryotes can be summarized as follows:

1. DNA unwinds at the origin of replication.

2. Helicase opens up the DNA-forming replication forks; these are

extended in both directions.

3. Single-strand binding proteins coat the DNA around the replication fork
to prevent rewinding of the DNA.

4. Topoisomerase binds at the region ahead of the replication fork to

prevent supercoiling (over-winding).

5. Primase synthesizes RNA primers complementary to the DNA strand.

6. DNA polymerase III starts adding nucleotides to the 3′-OH (sugar) end of
the primer.

7. Elongation of both the lagging and the leading strand continues. 73

Block 3 DNA-Blueprint of Life
8. RNA primers are removed and gaps are filled with DNA by DNA
polymerase I.

9. The gaps between the DNA fragments are sealed by DNA ligase.

During replication some problems are faced by eukaryotes. Some of these

have been listed below:

• The chromosomes are linear in eukaryotes and they possess more

amount of genetic material. A typical animal cell has 50 times more DNA
than the bacterium. e.g. E. coli has 4.6 million base pairs, humans have
3000 million base pairs.

• Eukaryotes show high level of packaging in the form of nucleosomes

(DNA wound around histones).

• DNA polymerases found in eukaryotes work slowly. More enzymes are

required to speed up the process of replication.

• The replication initiates at multiple sites scattered some 30 to 300 kb

apart.

Differences in DNA replication of prokaryotic and eukaryotic cells has been

summarized in the table given below (Table 11.3).

Table 11.3: Comparative account of DNA replication in prokaryotes and

eukaryotes.

DNA Replication in Prokaryotic Cells DNA Replication in Eukaryotic Cells

Occurs in the cytoplasm Occurs in the nucleus
There is a single origin of replication for There are multiple origins of replication
each DNA molecule.

Replication occurs at one point in each Replication at several points

DNA molecule. simultaneously in each chromosome.
Only one replication fork is formed in one Numerous replication bubbles are
DNA molecule. formed in one DNA molecule.
Both initiation and elongation is carried Initiation is carried out by DNA
out by DNA polymerase III polymerase α while elongation occurs
with the help of DNA polymerase δ and
ε.
DNA gyrase is required DNA gyrase is not required.
Replication is very fast i.e. about 2000 Replication is slow i.e. about 100
base pairs/nucleotides are added per nucleotides are added per second.
second.
The Okazaki fragments are very long The Okazaki fragments are short (100-
(1000-2000 nucleotides long) 200 nucleotides long)
RNA primer is removed by DNA RNA primer is removed by DNA
polymerase I. polymerase β.
DNA is circular, hence telomeres are not Telomeres present at the end of DNA are
replicated replicated
74
Unit 11 DNA Replication

SAQ 3
a) State whether these statements are ‘True’ or ‘False’.

i) The large DNA molecules in eukaryotes take more time to

replicate.

ii) More than two polymerases are used for replication of leading and
lagging strands at replication fork in eukaryotes.

iii) For faster DNA replication in eukaryotes, DNA synthesis is initiated

at multiple sites.

iv) Five forms of DNA polymerases are found in eukaryotes.

v) In eukaryotes, Polymerase δ is the only enzyme that shows 3ˊ 5ˊ

exonuclease activity required for proof reading.

b) Explain the role of telomerase in DNA replication.

11.6 SUMMARY
• DNA replication refers to duplication of DNA molecules resulting in
formation of copies of molecules during cell division DNA replication is
also referred as duplication.

• DNA replication can be conservative, dispersive or semi-conservative. In

conservative type, one molecule is conserved as such and a new copy
of DNA is synthesized from the old molecule. In dispersive type, the old
molecule disintegrates and two molecules would be synthesized, while in
semiconservative type of replication, the two strands separate from each
other, maintain their integrity and synthesize its complementary strand
from the pool of nucleotides.

• The prokaryotes show bidirectional, semiconservative mode of DNA

replication. DNA replication always begins at specific sites on the
bacterial chromosome called the origin. At this site the proteins bind and
initiate the process of replication. The replication proceeds outward from
the origin in both the directions. The pair of replicated segments come
together and joins at site called replication fork. At this site, two strands
get separated and nucleotides get incorporated into newly synthesized
complimentary strands. The two replication forks move in opposite
directions until they meet at a point across the circle from the origin
where the replication is terminated.

• DNA polymerases are the enzymes that synthesize new DNA strands.
The DNA strands serve as templates for the polymerization reaction.
The strand provides the necessary 3ˊ OH terminus called as primer. The
enzyme adds nucleotides to the 3ˊ hydroxyl terminus of an existing
strand. The enzyme DNA polymerase synthesizes DNA in a 5ˊ to 3ˊ
direction. 75
Block 3 DNA-Blueprint of Life
• The two strands are synthesized by different processes. One strand is
synthesized continuously, it is called the leading strand. The other strand
is synthesized discontinuously, it is called the lagging strand. Since one
strand is synthesized continuously and other discontinuously, the
replication is called as semidiscontinuous.

• DNA replication in eukaryotes is different from that in prokaryotes

because eukaryotes s have larger genomes and complex chromosomal
structures. Large DNA molecules in eukaryotes take more time to
replicate. Eukaryotic chromosomes contain multiple origins of replication.
Two or more polymerases are used for replication of leading and lagging
strands at replication fork.

• DNA polymerase III begins to add deoxyribonucleotides, initiating DNA

synthesis. DNA polymerase III efficiently synthesizes DNA in the
direction of replication fork i.e. in 5΄-3΄ direction with the template
showing 3΄-5΄ polarity. The leading strand is synthesized continuously
while the lagging strand is synthesized discontinuously to form Okazaki
fragments

• J. Cairns verified the semiconservative method of replication

photographically using autoradiography technique. The examining of
autoradiographs revealed that DNA of E. coli is circular and replicates
maintaining the integrity of the circle.

• Telomeres contain enzyme called telomerase which help to prevent the

shortening of chromosome ends. Telomerase simply extends the 3ˊ end
of the template strand. The unique feature of telomerase is that it
contains an inbuilt RNA template. Integral RNA component of
telomerases contain a sequence complementary to the repeat sequence
that makes it act as a template for a reverse transcriptase reaction.

11.7 TERMINAL QUESTIONS

1. Describe Meselson and Stahl experiment to explain semi conservative
mode of replication.

2. Rolling circle replication is noted in which organisms and how is it

different from other modes of DNA replication?

3. Write a short note on ‘end-replication” problem’.

4. Summarize the various steps involved in the process of DNA replication

in eukaryotes.

5. Compare the process of DNA replication in prokaryotes and eukaryotes.

11.8 ANSWERS
Self Assessment Questions
1. a) i) Refer to Section 11.2.

76 ii) Refer to Subsection 11.3.2.

Unit 11 DNA Replication
b) i) Refer to Subsection 11.2.1.

ii) Refer to Box 11.2.

iii) Refer to Subsection 11.3.2.

2. a) i) ori C

ii) unwinding

iii) Helicase

iv) negative supercoils

v) primer

vi) RNA segment

vii) DNA polymerases III

viii) proof reading

b) i) Refer to Subsection 11.4.3.

ii) Refer to Subsection 11.4.4.

iii) Refer to Subsection 11.4.5.

3. a) i) True; ii) True; iii) True; iv) False; v) False

b) Refer to Subsection 11.5.4.

Terminal Questions
1. Refer to Subsection 11.2.1.

2. Refer to Subsection 11.3.3.

3. Refer to Subsection 11.5.4.

4. Refer to Section 11.5.

5. Refer to Table 11.3.

Acknowledgment for Figures

Fig. 11.1 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fi1.w
p.com%2Fmicrobiologyidea.com%
Fig. 11.3 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fi.pini
mg.com%2Foriginals
Fig. 11.4 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fww
w.mun.ca%2Fbiology%2Fdesmid%2Fbrian%2FBIOL2060%2FB
IOL2060-19%2F19_09.jpg 77
Block 3 DNA-Blueprint of Life
Fig. 11.5 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fima
ge.slidesharecdn.com%2Freplication-111109072715-
phpapp01%2F95%2Freplication-18-728.jpg
Fig. 11.6 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fslide
todoc.com%2Fpresentation_image%2F499792226df80c6a61de
05edc6af2e09%2Fimage-21.jpg
Fig. 11.7 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fmicr
obiologynotes.org%2Fwp-content%2Fuploads%2F2019%2F
Fig. 11.8 : Source:
https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fww
w.nature.com%2Fscitable%2Fcontent%2Fne0000

78