"DNA REPLICATION IN PROKARYOTES AND

EUKARYOTES"

Submitted to
DEPARTMENT OF ZOOLOGY
Shia P.G. College
LUCKNOW

By
___________
Father's Name- _______________
Roll No- ________________
BSc. Semester VI
SESSION-2023-2024

SUPERVISORS

_____________________ ____________________
Department of Zoology Associate professor
Department of Zoology

ACKNOWLEDGEMENT
I would like to express my special gratitude to all those who gave me this wonderful

opportunity to work on this amazing project "" DNA REPLICATION IN

PROKARYOTES AND EUKARYOTES"" Through which I gain so much knowledge.

I am also very thankful to my supervisors, DR NAZEER HAIDER ZAIDI AND DR

MOHD ZAHID RIZVI for providing me proper guidance regarding my project. I would

also like to thank our H.O.D of Department of Zoology Prof. Syed Rais Haider Sir and all

faculty members. I would also like to thank our respected Principal sir Prof. S Shabihe Raza

Baqri.

Last but not the least I would like to thank my parents who helped me in shaping up my

project on time.

Thankyou.

Date:

Place: Lucknow

_____________
B.Sc. VI Semester (ZOOLOGY)
Roll No- ______________
 TABLE OF CONTENT

Chapter 1: Introduction

Chapter 2: Review of Literature

Chapter 3: Conclusion

Chapter 4: Summary

References
 INTRODUCTION

DNA replication is a fundamental process in the perpetuation of life, ensuring

the faithful transmission of genetic information from parent to offspring. It is

essential for cell division, growth, and the maintenance of genetic integrity. The

intricate mechanisms governing DNA replication have been extensively studied

in both prokaryotic and eukaryotic organisms, revealing remarkable similarities

as well as notable differences between these two domains of life.

In prokaryotes, such as bacteria and archaea, DNA replication occurs in a

relatively straightforward manner due to the simplicity of their genetic material,

typically consisting of a single circular chromosome. Key players in prokaryotic

DNA replication include the DNA polymerase enzyme, helicase, primase, and

DNA ligase, which coordinate to unwind the DNA double helix, synthesize new

DNA strands, and join them together.

Eukaryotic DNA replication, on the other hand, is more complex due to the

presence of multiple linear chromosomes housed within the nucleus. This

necessitates additional layers of regulation and coordination to ensure accurate

replication of the genome. Eukaryotic DNA replication involves a larger

number of proteins and regulatory factors compared to prokaryotes, including

multiple DNA polymerases with specialized functions, origin recognition

complexes, and chromatin remodeling proteins.

Understanding the mechanisms of DNA replication in both prokaryotes and

eukaryotes is of paramount importance for various fields of biology, including

genetics, molecular biology, and biotechnology. Insights gained from studying

DNA replication have far-reaching implications, ranging from elucidating the

molecular basis of genetic diseases to informing the development of novel

therapeutic interventions and biotechnological applications.

 REVIEW OF LITERATURE

DNA replication is an essential property of the genetic material. DNA replication is the

process by which DNA makes exact and accurate copy of itself. So, by replication from

single double stranded DNA molecule 2 double stranded DNA molecules are formed.

It occurs during S-phase of the interphase prior to the cell division, so when the cell divides

each daughter cell will get equal amount of the genetic material. Approximately 20 or more

enzymes and proteins are during replication.

Watson and Crick’s model for DNA replication

The double helix model of DNA molecule of Watson and Crick beautifully embodied a built-

in template system for self-replication or autocatalytic function. Because of the specificity of

base pairing, the sequence of base along one chain automatically determines the base

sequence along the other. Thus, each chain of the double helix can serve as template for the

synthesis of the other. For the replication of DNA molecule, Watson and Crick proposed that

replication involved the disruption of hydrogen bonds followed by a rotation and separation

of the two polynucleotide strands. Each purine and pyrimidine base of each polynucleotide

strand is thought to attract a complementary free nucleotide polymerization in cell and to hold

it in place means of the specific hydrogen bonds. Once held in place on the parent template

chain, the free nucleotides were sewn together by formation of the phosphate-di-ester bonds

that linked adjacent deoxyribose residues, forming a new polynucleotide molecule of

predetermined base sequence. Thus, two double helical molecules, identical with each other,

are formed.

Replication Models:

There are three models which explain the accurate replication of DNA. These are: (i)

dispersive replication, (ii) conservative replication, and (iii) semiconservative replication

These are explained as follows:

(i) Conservative Replication: According to this model of DNA replication two DNA

molecules are formed from parental DNA. One copy has both parental strands and the other

contains both newly synthesized strands. This method is also not accepted as there is no

experimental proof in support of this model.

(ii) Semiconservative Replication: This model of DNA replication was proposed by

Watson and Crick. According to this model of DNA replication, both strands of parental

DNA separate from each other. Each old strand synthesizes a new strand. Thus each of the

two resulting DNA molecules has one parental and one new strand. This model of DNA

replication is universally accepted because there are several evidences in support of this

mode. The mode of DNA replication is semi-conservative. The experimental evidence was

given by Meselson and Stahl (1958).

(iii) Dispersive Replication: According to this model of replication the two strands of

parental DNA break at several points resulting in several pieces of DNA. Each piece

replicates and pieces are reunited randomly, resulting in formation of two copies of DNA

from single copy. The new DNA molecules are hybrids which have new and old DNA in

patches. This method of DNA replication is not accepted as it could not be proved

experimentally.
Meselson-Stahl Experiment:

The experimental for semiconservative replication of DNA was first demonstrated by

Mathew Meselson and Franklin Stahl in 1958. They grew cells of the bacterium Escherichia
15 14
coli on a medium that contained N (a heavy isotope of N) in the form of ammonium

chloride (NH4C1).

15
The cells of E. coli were grown for 14 cell generation so that the cells used the N to

synthesise bases which were then incorporated into DNA. DNA having 15N has a detectable

higher density than that having 14N.

Therefore, they are called heavy and light DNA, respectively. Heavy and light DNA can be

separated readily through equilibrium density gradient centrifugation where they form

distinct band in the centrifuge tube.

14
The cells of E. coli were then sub-cultured and grown to a medium containing normal N.
14
The transfer of cell to medium containing normal N was followed by extraction of DNA

from cells after zero, one, two, three etc. cell generations (one cell generation represents the

time during which all the cells undergo one cell division).

The extracted DNAs in each cell generation were analysed in cesium chloride (CsCl) density

gradient.
 15
DNA that containing N in both strand (heavy DNA) forms a band in the CsCl density

gradient at a higher density position than that contains 14N in both strand (light DNA). After

one generation of growth in 14N medium, the DNA bands at an intermediate (hybrid DNA)

density.
15 14
Such hybrid DNA contains N in one strand and N in the other strand. After two

generations of growth in 14N medium, half of the DNA bands are seen at the hybrid density

and half band at light density.

It may be pointed out that according to the conservative mode of replication, no intermediate

band will appear after one generation. Only heavy and light bands would be formed.

Similarly, the dispersive mode of replication would make only one band having identical

density. No light or intermediate bands would be formed.

Therefore, the result of Meselson and Stahl’s experiment clearly explains the

semiconservative mode of DNA replication, while the expectation of other mode of

replication (conservative, dispersive) are not fulfilled.

 DNA replication in prokaryotes-

In bacteria, chromosome is circular made up of double stranded DNA molecule.

DNA replication in E. coli and other prokaryotes occurs by a semiconservative mechanism

in which the strands of a DNA double helix separate and a new complementary strand of

DNA is synthesized on each of the two parental strands which are the template strands

forming the two dsDNA molecules, each with one strand from the Parent DNA molecule and

one newly synthesized strand.

Basic requirements for DNA replication

1. 4 dNTPs : dATP, dTTP, dGTP, dCTP (2’deoxyribonucleoside 5’-triphosphates),

2. DNA template

3. RNA primer – small complementary piece of mRNA

4. Enzymes

5. Proteins

6. Mg2+ (optimizes DNA polymerase activity)

Enzymes involved in DNA synthesis-

1. DNA Polymerase: DNA polymerase is the chief enzyme of DNA replication. DNA

polymerase activity was discovered by Kornberg in 1956; this activity was due to DNA

polymerase I. E. coli has four more enzymes, DNA polymerase II, III, IV and V; DNA

polymerase III (Pol III) is concerned with DNA replication, while the remaining four

enzymes are involved in DNA repair. All DNA polymerases require the following: (1) A

template DNA strand, (2) A short primer (either RNA or DNA), and (3) A free 3′ -OH in the

primer. They add one nucleotide at a time to the free 3′ -OH of the primer, and extend the

primer chain in 5′ → 3′ direction.

A. DNA Polymerase I: DNA polymerase I enzyme provides the major part of activity in
 E. coli. It is chiefly a DNA repair enzyme, and is used for in vitro DNA replication.

This enzyme has the following three activities:

(i) The 5′ → 3′ polymerase activity is responsible for primer extension or DNA synthesis.

(ii) The 5′ → 3′ exonuclease activity is involved in excision of DNA strands during DNA

repair; it removes ~ 10 bases at a time. An exonuclease digests nucleic acids (here DNA)

from one end, and it does not cut DNA internally.

(iii) The 3′ → 5′ exonuclease activity is responsible for proof-reading.

In this case, only one nucleotide is removed at a time. The polymerase action does commit

errors in DNA synthesis. DNA polymerase is known to scrutinize the new bases added to the

growing chain and to delete or remove the wrong bases; this is called proof-reading. Proof-

reading activity reduces errors in replication by over 100 – fold.

DNA polymerase I is encoded by gene polA, has a single polypeptide, and can initiate

replication in vitro at a nick in a DNA duplex. It can be cleaved by proteolytic treatments into

a large and a small fragments. This large fragment, called Klenow fragment, lacks 5′ → 3′

exonuclease activity and is used for in vitro DNA replication.

B. DNA Polymerase II: DNA polymerase II enzyme functions in DNA-repair. It has 5′

→ 3′ polymerase and 3′ → 5′ exonuclease activities, and uses as template only such DNA

duplexes that have short gaps.

C. DNA Polymerase III: DNA polymerase III enzyme is responsible for DNA

replication in vivo. It has 5’→ 3′ polymerase and 3’→ 5′ exonuclease activities. It

catalyzes DNA synthesis at very high rates, e.g., 15,000 bases/min at 37°C. It is composed

of several subunits. A DNA polymerase molecule has the following 4 functional sites

involved in polymerase activity.

  Template site binds the strand serving as template

 during replication.

 Primer site binds to the primer used for DNA replication.

 Primer terminus site binds only to such primers that have free 3′ -OH.

 The nucleotide triphosphate site binds to the deoxynucleotide 5′-triphosphate that is

complementary to the corresponding nucleotide of the template. It also catalyzes the

formation of phosphodiester bond between the 5′ phosphate of this nucleotide and the

3′ -OH of the terminal primer nucleotide.

Properties of DNA Polymerases in Prokaryotes

Parameter DNA Pol I DNA Pol II DNA Pol II

(Kornberg enzyme)

Nature 1 polypeptide 4 polypeptides 10polypeptides

No. of copies/cell 300~400 About 40 10-20

Polymerization direction 5’ 3’ 5’ 3’ 5’ 3’

Exonuclease activity 3’ 5’ , 5’ 3’ 3’ 5’ 3’5’

Mol. wt.(daltons) 1,09,000 1,20,000 Core 1,30,000

Structural genes pol A pol B dna E, dna Q, dna H (foe

 core)

Functions Principal repair, Error prone Principal Polymerization,

Primer exicision, repair Recognition of primer, Poof

Polymerization. Reading, Repair, Speed &

efficiency of

replication.

2. Primase: This enzyme activity catalyzes the synthesis of RNA primers to initiate

DNA replication. In E. coli, DnaG functions as primase. But in eukaryotes, DNA

polymerase α provides this function.

3. Polynucleotide Ligase or DNA ligases: DNA ligase enzymes are capable of catalyzing

phosphodiester bond formation between free 3´-OH and free 5´-P groups of a nick of DNA

which is created by endonuclease enzyme, thereby restoring an intact DNA duplex. Many

DNA ligases have already been discovered. The ligase enzyme from E.coli requires the

presence of oxidized nicotinamide adenine dinucleotide (NAD+) a cofactor, whereas the

ligase enzyme specified by T4 bacteriophage requires ATP to bring about the joining

reaction.

4. Nuclease enzymes: The nuclease enzymes act to hydrolyze or break down a

polynucleotide chain into its component nucleotides. A polynucleotide is held together

by 3´, 5´
phosphodiester bonds and a nuclease enzyme will attack either the 3´ or the 5´ end of

this linkage. The nuclease enzymes may be of the following two kinds:

(a) Exonuclease enzymes: A nuclease enzyme which begins its attack from a free end of

a polynucleotide is called exonuclease. Therefore, depending on the specificity of the

enzyme, an exonuclease will either begin at a free 3'-OH end of a polynucleotide and

progressively cleave the bonds on the 3'-OH side of the phosphodiester backbone or it will

begin at a free 5'-P end and digest the polynucleotide in a 5' → 3' direction. In’ both cases

the enzyme travels along the chain in a stepwise manner, liberating single nucleoside

monophosphate molecules and eventually digesting the entire polymer.

(b) Endonuclease enzyme: Endonuclease enzyme also attacks one of the two sides of

phosphodiester linkages, but they react only with those bonds that occur within the interior

of a polynucleotide chain. If the polynucleotide chain is single stranded (e.g., viral DNA),

such an attack will obviously cut the chain into two pieces. If, however, the polynucleotide

strand is a member of a DNA helix (e.g., prokaryotic and eukaryotic DNA), a single

endonucleolytic cut will create a nick in the helix; the helix remains in one piece but it now

possesses a gap that contains two tree ends, which can serve as substrates for exonucleases.

5. DNA helicases: DNA helicases are ATP dependent unwinding enzymes which

promote separation of the two parental strands by disrupting hydrogen bonds.

Hydrolysis of ATP drives the reaction. Unwinding of the template DNA helix at a replication

fork could in principle be catalyzed by two DNA helicases, acting in concert, one running

along the leading strand and the other along the lagging strand. Among E. coli helicases are

the DnaB protein and the Rep protein. The Rep protein may help to unwind the double

helix ahead of the polymerase.

6. Topoisomerases (DNA gyrases): The action of helicases during DNA replication

generates twists in the circular DNA that need to be removed to allow replication to continue.

Circular DNA can be twisted and coiled, much like the extra coils that can be introduced into

a rubber band. This supercoiling can be created or relaxed by enzymes termed

topoisomerases, an example of which is DNA gyrase. Topoisomerases can also induce or

remove knots, or links in a chain. There are two basic types of isomerases. Type I enzymes

induce a single-stranded break into the DNA duplex. Type II enzymes cause a break in

both strands. In E. coli, topo I and topo III are examples of type I enzymes, whereas

gyrase is an example of a type II enzyme. The breaks introduced by both these enzymes

serve as swivels. These allows the 2 strands of DNA to rotate freely around each other &

preventing over winding of DNA.

7. DNA Primase- It is a product of dna G. Its function is to synthesis mRNA primer. In

bacteria, two different enzymes are known to synthesize primer RNA oligonucleotides –

RNA polymerase (on the leading strand) and DNA primase (on the lagging strand).

8. RibonucleaseH ( RNaseH)- It is non-sequence specific endonuclease that catalyze the

cleavage of RNA in an RNA/DNA substrate via a hydrolytic mechanism or exision of

mRNA primer after its use. Members of the RNase H family can be found in nearly all

organisms, from bacteria to eukaryotes.

Single-Strand Binding (SSB) Protein: (also called Helix-destabilizing strand ) SSB

protein binds to single-stranded DNA, and prevents it from forming duplex DNA or

secondary structures. SSB binds as a monomer, but it binds cooperatively in that binding of

one SSB molecule facilitates binding of more SSB monomers to the same DNA strand. E.

coli SSB is a tetramer.

Origin of Replication: (ori): The replication of DNA (Pro/Eukaryotic) starts at a specific

or unique sequence of nitrogenous bases in the DNA mol. called Origin of replication. In
prokaryotes there is a single origin of replication where as in eukaryotes it is multi

original.

Roles of RNA Primers in DNA Replication: Polymerases can extend a chain but cannot

start a chain. Therefore, as already mentioned, DNA synthesis must first be initiated with a

primer (short oligonucleotide) , that generates a segment of duplex DNA. Primase

synthesizes a short (approximately 30 bp long) stretch of RNA complementary to a specific

region of the DNA. The RNA chain is then extended with DNA by DNA polymerase. The

primers are about 10 nucleotides long in eukaryotes and they are made at intervals on the

lagging strand where they re elongated by the DNA polymerase enzyme to begin each

okazaki fragment. These RNA primers are later excised and filled with DNA with the help of

DNA repair system in eukayrotes (or DNA polymerase I in E.coli). In bacteria, two

different enzymes are known to synthesize primer RNA oligonucleotides – RNA

polymerase (on the leading strand) and DNA primase (on the lagging strand).

Replicons: DNA replication in prokaryotes and eukaryotes is attained in discrete units,

called replicons. The number of replicons may vary in a genome from one in bacteria

(E.coli) and 500 in yeast to several thousands in plants and animals. For example, in the

E. coli there is single replicon with the origin, identified as a genetic locus ori C (245 bp).

The origin is A: T rich, a feature that is related to unwinding of DNA to initiate replication. In

E. coli, there are also termination sites (ter A-F), each consisting of ~ 23 bp. The process of

termination of DNA replication requires the product of tus gene (Tus protein or TBP, i.e., ter

binding protein) which recognizes ter or termination sites.

Replisome and Primosome: The DNA polymerases, RNA primases and helicases may be

associated with one another to form a multienzyme complex — the replisome that carries out

the synthesis of leading and lagging strands in a coordinated fashion. Such a complex would
be highly processive and assure rapid replication of the DNA. Likewise, the proteins at a

replication fork cooperate to form a replication machine, e.g., the primase molecule is linked

directly to the DNA helicase to form a unit on the lagging strand called a primosome which

moves with the fork, synthesizing RNA primers as it moves.

Prokaryotic & Eukaryotic Replicons

Molecular mechanism of Replication in Prokaryotes:

Replication is an enzymatic process in which synthesis of a daughter duplex DNA molecule,

identical to the parental duplex DNA occurs. Rate of replication in E. coli (prokaryotic cell)

is 1500 nucleotides /second. To complete replication of whole E. coli genome it takes 40

minutes. In prokaryotic circular DNA only one replication fork is present but in eukaryotic

DNA several replication forks are present.

The synthesis or replication of DNA molecule can be divided into three stages
 I) Unwinding, II) Biosynthesis of new strands, III) Winding

I) Unwinding: In order for duplex DNA to function as a template during replication local

separation of the 2 strands (denaturation) at a specific sequence or initiation point called

origin of replication (OriC) is required. It is the point where DNA open up and form open complex

(replication bubble) leading to the formation of pre-priming complex to initiate replication process.

The Ori C site consists of 245 base pairs, of which three of 13 base pair sequence (13mers)

are highly conserved in many bacteria and forms the consensus sequences

(GATCTNTTNTTTT). Close to Ori C site, there are four of 9 base pair sequences (9mers)

each (TTATCCACA).

A single complex of 30 molecules of Dna A protein recognizes and binds up to four 9bp

repeats in OriC and then successively melt three tandemly repeated 13bp segments in the

presence of ATP and bacterial histone like proteins (HU). The Dna A protein then guides a

Dna B - Dna C complex into the melted region. The Dna C is subsequently released. Dna B

further unwinds open complex. In the presence of Gyrase and SSBP, helicases (Dna A,

DnaB, DnaC) further unwinds the DNA in both directions so as to permit entry of primase

and RNA polymerase.

II) Biosynthesis of new strands:

1. Once the helicase have unwound the parental DNA at an origin, then RNA polymerase

forms primer for leading strand synthesis while primase in the form of primosome synthesis

primer for lagging strand synthesis. Primer is the small piece of RNA which is
complementary to DNA bind to template to which then successive complementary bases are

added. To the above complex, DNA polymerase - III will bind and forms replisome. This

RNA primres are later removed and replaced by DNA fragments.

2. The DNA region at which all these proteins come together to carry out synthesis of new

strand is called replication fork. Single strand DNA binding proteins (SSBP) binds to

ssDNA that prevents recoiling of separated DNA strands and stabilizes them so that they can

act as a template.

3. Conversion of dNTPS into nucleotides- dNTPs pair with complementary bases of

DNA template, on pairing dNTPs sets free pyrophosphates (p-p) & change to

deoxyribonucleotides. The enzyme pyrophosphatase hydrolyses the p-p into inorganic

phosphates (pi) & releases energy.

4. Polymerization of DNA strands- The energy released by conversion of dNTPs into

nucleotides is utilized in polymerization of new strands, catalyzed by DNA polymerase along

with Mn++ & Mg++ ions.

5. Synthesis of leading strand- The major complications in DNA replications are

antiparallel nature of 2 DNA strands (5’3’ & 3’5’) & ability of DNA polymerase to adds

nucleotides to the growing new strands only in 5’3’direction.

So, the new strand on 3’—5’ template grows towards replication fork in 5’3’ direction with

single RNA primer by addition of nucleotides to 3’ end. This strand is synthesized without

interruption, therefore called leading strand. The reaction catalyzed by DNA pol-III is very

fast. The enzyme is much more active than DNA pol - I and can add 9000 nucleotides/

minute at 370C. The RNA primer that was initially added by RNA polymerase is degraded

by RNase.
6. Synthesis of lagging strand- In contrast, the new strand with 3’5’ polarity (i.e., on

5’3’ template) elongates away from the replication fork. It is synthesized in discontinuous

complex fashion on the 3'5' template is called lagging strand. It involves synthesis of series

of discontinuous short segments of nucleotides called Okazaki fragments. Synthesis of

Okazaki fragments involves the synthesis of RNA primer by primosome, 1000-2000

nucleotides are added by DNA pol-III. When the Okazaki fragment synthesis was completed

up to RNA primer, then RNA primer was removed by DNA pol - I using its 5'3'

exonuclease activity. The gap created by the removal of primer, is filled up by DNA pol - I

using the 3'-OH of nearby Okazaki fragment by its polymerizing activity. Finally, the nick
existing between the fragments are sealed by DNA ligase which catalyze the formation of

phosphodiester bond between a 3'-OH at the end of one strand and a 5' - phosphate at the

other end of another fragment. The enzyme requires NAD for during this reaction.

Since synthesis of leading strand is continuous & lagging strand is discontinuous, the

overall replication of DNA is said to be semi-discontinuous.

7. Proof reading: DNA pol. is also responsible for proof reading at replication process. If

any incorrect nucleotide get incorporates into the DNA strand 3’5’ exonuclease activity of

DNA pol is triggered at 3’ end of growing strand, removing the incorrect base & again

polymerization is resumed.

7. Termination- Termination occurs when the two replicating forks meet each other on the

opposite side of circular E.coli DNA. Termination site, Ter A terminates the counter

clockwise moving fork while Ter C terminates the clockwise moving forks. Termination at

these sites are possible because, at these sites tus protein (Termination utilizing substance)

will bound to Dna B protein and inhibits its helicase activity. After the complete synthesis,

two duplex DNA are found to be catenated (knotted). This catenation removed by the action

of Topoisomerase IV. Finally, from single parental duplex DNA, two progeny duplex DNA

synthesized.

III. Winding: At the end one new strand gets coiled with one old strand by removal of SSBP

restoring the double helical structure. So, both the daughter molecules get segregated.
Rolling Circle Replication:

In many bacteriophages and also in bacterial conjugation, a circular double-stranded DNA

molecule gives rise to a linear DNA by rolling circle replication. In this type, a break (nick) is

produced in one strand to expose a 3′-OH end and a 5′-(P) end. A replication fork is produced

by a helicase and associated SSB proteins. The 5′-(P) end is displaced and it acts as the

template for synthesis of the lagging strand in Okazaki fragments in the 5’—>3′ direction.

The exposed 3′- OH end of the nicked strand can add nucleotides from precursors to elongate

the polynucleotide chain using the intact DNA strand as template. Rolling circle implication

results in a sigma (σ) configuration consisting of a rolling circle and a linear branch. In some

cases, because there is no termination point, synthesis often continues beyond a single circle

unit, producing concatamers i.e., a series of linked chains, of several circle lengths, which are

then processed by recombination to yield normal length circles. Rolling circle replication

also occurs in F-plasmids of conjugating bacteria, like E. coli. Rolling circle replication

occurs also in single-stranded DNA phages, like φX174 which reproduces through a double-

stranded intermediate.
EUKARYOTIC DNA REPLICATION (Replication of Linear DNA):

The replication in eukaryotic chromosome is more complex in as much as they are linear &

usually much larger than bacterial chromosome. Eukaryotic replication occurs during s-phase

(interpahse) of cell cycle. As a result of replication, each chromosome consists of 2 identical

chromatids joined together, which are then segregates into daughter cells by the mitosis. In

the G1 phase the cell prepares itself for DNA replication. In yeast a checkpoint known as

START decides whether the cell should continue into the S phase. In mammalian cells this

check point is known as the G1 checkpoint. Unless the cell has grown large enough and the

environment is favourable it will stay in the G 1 phase. Another checkpoint called G2

checkpoint occurs just before the M phase. The cell cannot enter mitosis unless all the DNA

has replicated. A third checkpoint occurs during the M phase. Chromosomes must be

attached to the mitotic spindle fibres in order to trigger the separation of chromatids and the

completion of mitosis. Proteins known as cyclins and enzymes known as cyclin-dependent

kinases (Cdks) are involved in the in the regulatory events that occur at the checkpoints.

However at the molecular level, the replication of DNA in eukaryotes is quite similar to that

of prokaryotes regardless of complexity of its genome. It appears to involve same enzymes

and mechanisms as in prokaryotes. The eukaryotic DNA polymerase have the same absolute

requirements for template & primer as prokaryotic polymerase. Eukaryotic DNA replication

is also semiconservative & semi discontinuous. Replication usually occurs only one time in a

cell.
Eukaryotic DNA Polymerases:

Parameter s Polymerase Polymerase Polymerase Polymerase  Polymerase 

 (alpha)  (beta)  (gamma) (delta) (epsilon)

Nuclear
Cytoplasmic
/small
polymerase
polymerase

Location Cytoplasm, Nucleus Nucleus Nucleus Nucleus

Nucleus

Occurrenc e Only in Mammalian Mammalian Mammalian

All eukaryotic vertebrate cells cells cells with RfC Hela cells,

cells & PCNA Budding Yeast

cells

Functions *Mitochon- *Replication of *Nuclear DNA

* Nuclear *Nuclear DNA
Repair drial DNA Lagging replication/Re
DNA Repair

*As Primase *No proof Replication strand (Nuclear), pair

Reading *3’5’ Proof * 3’5’ Proof * 3’5’ Proof

*No proof
Reading Reading Reading
Reading

1. DNA polymerase α (pol α): DNA polymerase α associated with enzyme Primase, forms

RNA primer which are 8-10 nucleotide long. Later DNA polymerase α elongates this RNA

primer by more 20 nucleotides and then leaves the place. Like all DNA polymerases,

replicates DNA by extending a primer in the 5’  3’ direction under the direction of a ssDNA

template. This enzyme has no exonuclease activity and therefore cannot proofread its

polymerization product. Pol α is only moderately processive (polymerizing ~100 nucleotides

at a time). It is found in both cytoplasm and nucleus. It lacks proof reading activity.
2. DNA polymerase β (pol β): occurs exclusively in the vertebrates. It is found in very

small amount, mostly responsible for DNA repair. It lacks proof reading activity.

3. DNA polymerase γ (pol γ): occurs exclusively in the mitochondrion, where it

presumably replicates the mitochondrial genome. Chloroplasts contain a similar enzyme. It

shows 3’5’ Proof Reading activity.

4. DNA polymerase δ: DNA polymerase δ helps the synthesis of DNA on lagging strand.

On the lagging strand multiple RNA primers are required. On the lagging strand, DNA

polymerase δ synthesizes small fragments of DNA called Okazaki fragments. It shows 3’5’

Proof Reading activity. DNA polymerase δ (pol δ) does not associate with a primase and

contains exonuclease activity. It can replicate the entire length of a template DNA, but only

when it is in complex with protein named proliferating cell nuclear antigen (PCNA).

5. DNA polymerase ε: DNA polymerase ε synthesis nucleotides on the leading strand. It

will continuously add nucleotides leading to continuous process of replication. Thus it will

require only one RNA primer at the beginning. It shows 3’5’ Proof Reading activity.

Molecular mechanism of DNA replication in eukaryotes

The sequential steps involved in DNA replication of eukaryotes are the same as for

prokaryotes.

Initiation:

1. Each eukaryotic chromosome is one linear DNA double helix, and on an average is ~10 8

base pairs long. Instead of single origin of replication, as in bacteria eukaryotic chromosome

have many origins for each chromosome in keeping with their much larger size. Replication

is bidirectional and since here are many sites of origin, eventually each replication fork runs

into the adjacent origin of replication.

2. The stretch of DNA from the origin of replication to the two termini of replication

(where adjacent replication forks fuse) is called a replicon or a replication unit.

3. There are a large no. of replicons in each eukaryotic chromosome. Each replicon has

about 50,000 – 3,00,000 nucleotides pairs. All replicons do not replicate simultaneously.

Cluster of 20

– 80 replicons are activated together.

4. Each origin of replication consists of about 150 bps. These autonomously replicating

sequences (ARS) are called replicators. Initiation of replication in all eukaryotes requires a

protein called the Origin Recognition Complex (ORC).

5. Separation of 2 strands (denaturation) takes place as in prokaryotes involving

Topoisomerase & proteins similar to the SSB proteins of prokaryotes also get attached to the

denatured eukaryotic DNA (at least in mammals) to prevent the formation of hairpin loops.

Replication bubble is formed due to separation of 2 strands at origin of replication.

6. It is then followed by joining of the three eukaryotic DNA polymerases and a number of

other proteins required for their recruitment. Interestingly, the polymerases assemble at the

origin in a particular order. DNA Pol and associate first, followed by DNA Pol

/primase. This order ensures that all three DNA polymerases are present at the origin

prior to the synthesis of the first RNA primer (by DNA Pol /primase).

Once present at the origin, DNA Pol /primase synthesizes an RNA primer and

briefly extends it. Thus initiation of replication started. Elongation: The resulting primer-

template junction is recognized by the replication factor C (RF-C), & PCNA at these

sites. Either DNA Pol or recognizes this primer and begins leading strand

synthesis. After a period of DNA unwinding, DNA Pol /primase synthesizes

additional primers, which allow the initiation of lagging strand DNA synthesis by either

DNA
Pol . Mostly, Pol is used for leading strand and Pol is used for lagging strand

synthesis. DNA Pol

Pol I in prokaryotes. SSB like activity was played by replication protein A (RP A) which is

denoted as accessory factors during replication.

Origin & initiation of replication in eukaryotes (Formation of replication bubble)

Formation of leading & lagging strands in eukaryotes (Elongation)

Termination: When the replication forks meet each other, then termination occurs. It will

result in the formation of two duplex DNA. Even though replication terminated, 5’ end of

telomeric part of the newly synthesized DNA found to have shorter DNA strand than the

template parent strand. This shortage corrected by the action of telomerase enzyme and then

only the actual replication completed.

Telomerase Function: In Linear eukaryotic chromosome, once the first primer on each

strand is remove, then it appears that there is no way to fill in the gaps, since DNA cannot be

extended in the 3'5' direction and there is no 3' end upstream available as there would be in

a circular DNA. If this were actually the situation, the DNA strand would get shorter every

time they replicated and genes would be lost forever.

Elizabeth Blackburn and her colleagues have provided the answer to fill up the gaps with the

help of enzyme telomerase. So, that the genes at the ends, are conserved. Telomerase is a

ribonucleoprotein (RNP) i.e. it has RNA with repetitive sequence. Repetitive sequence

varies depending upon the species example Tetrahymena thermophilia RNA has AACCCC

sequence and in Oxytrica it has AAAACCCC. Telomerase otherwise known as modified

Reverse Transcriptase. In human, the RNA template contains AAUCCC repeats. This

enzyme was also known as telomere terminal transferase.

The 3'-end of the lagging strand template basepairs with a unique region of the telomerase

associated RNA. Hybridization facilitated by the match between the sequence at the 3'-end of

telomere and the sequence at the 3'-end of the RNA. The telomerase catalytic site then adds

deoxy ribonucleotides using RNA molecule as a template, this reverse transcription proceeds

to position 35 of the RNA template. Telomerase then translocates to the new 3'-end by

pairing with RNA template and it continues reverse transcription. When the G-rich strand

sufficiently long, Primase can make an RNA primer, complementary to the 3'-end of the

telomere's G-rich strand. DNA polymerase uses the newly made primer to prime synthesis of
DNA to fill in the remaining gap on the progeny DNA. The primer is removed and the nick

between fragments sealed by DNA ligase.

Assembly of new DNA into nucleosomes:

Since eukaryotic DNA is complexed with histones in the nucleosome, the replication of DNA

is accompanied by the doubling of the histone complement followed by assembly into

nucleosomes.
 REPLICATION IN PROKARYOTES

Replication is an enzymatic process in which synthesis of a daughter or progeny

duplex DNA molecule, identical to the parental duplex DNA occurs. Rate of
replication in E.Coli (prokaryotic cell) is 1500 nucleotides per second. To complete
replication of whole E.Coli genome it takes 40 minutes.

The synthesis or replication of DNA molecule can be divided into three stages

I) Initiation (Formation of Replisome)

II) Elongation (Initiation of synthesis and elongation)

III) Termination

I) Initiation

The replication begins at a specific initiation point called Ori C point or

replicon. (Replicon: It is a unit of the genome in which DNA is replicated; it contains
an origin for initiation of replication) It is the point of DNA open up and form open
complex leading to the formation of prepriming complex to initiate replication
process.

The Ori C site consists of 245 basepairs, of which three of 13 basepair sequence are
highly conserved in many bacteria and forms the consensus sequences
(GATCTNTTNTTTT). Close to OriC site, there are four of 9 basepair sequences
each (TTATCCACA).
The sequence of reactions in the initiation process is as follows:

a) Dna A protein recognizes and binds up to four 9 bp repeats in Ori C to form a

complex of negatively supercoiled Ori C DNA wrapped around a central core of Dna
A protein monomers. This process requires the presence of the histone like HU or 1
HC proteins to facility DNA bending.

b) Once the four 9 bp repeats are

occupied 20 – 40 additional Dna
A monomers bind, so that entire
Ori C region is complexes with
Dna A protein.

c) The resulting complex

resembles a nucleosome with
negatively supercoiled Ori C
DNA wrapped around a DNA
core.

d) HU, a histone like protein

prevents nonspecific initiation at
sites other than the Ori C.

e) Dna A protein subunits then

successively melt three tandemly
repeated 13 bp segments in the
presence of ATP, which results in
the formation of 45 bp open
complex.
f) The Dna A protein then guides a
Dna B - Dna C complex into the
melted region to form a so called
prepriming complex. The Dna C is
subsequently released. Dna B
further unwinds open complex to
form prepriming complex.

g) DNA gyrase, single stranded

binding protein (SSB), Rep protein
and Helicase - II are bound to
prepriming complex and now
complex is called as priming
complex.

h) In the presence of gyrase and

SSB, helicases further unwinds the
DNA in both directions so as to
permit entry of primase and RNA
polymerase. Then RNA polymerase
forms primer for leading strand
synthesis while primase in the form
of primosome synthesis primer for
lagging strand synthesis.

f) To the above complex, DNA

polymerase - III will bind and forms
replisome.
REPLISOME: It is the multiprotein
structure that assembles at the bacterial
replicating fork to undertake synthesis of
DNA. It contains DNA polymerase and other
enzymes.

II) ELONGATION

Now the stage is set for the initiation of synthesis and the elongation to proceed. But
this occurs in two mechanistically different pathways in the 5'-->3' template strand
and 3'-->5' template strand.

Initiation of synthesis and Elongation on the 5'-->3' template (If replication fork
moves in 3'-->5' direction)

Synthesis of leading strand

 The DNA daughter strand that is synthesized continuously on 5'-->3' template

is called leading strand.
 Leading strand synthesis begins with the synthesis of RNA primer (10 -60
nucleotides) by primase (DNA g protein)
 DNA pol-III synthesizes DNA by adding 5'-P of deoxynucleotide to 3'-OH
group of the already presenting fragment.
 Thus chain grows in 5'-->3' direction. The reaction catalyzed by DNA pol-III
is very fast. The enzyme is much more active than DNA pol - I and can add
9000 nucleotides per minute at 37*C. The RNA primer that was initially added
by RNA polymerase is degraded by RNase.
Synthesis of lagging strand

Initiation of synthesis and Elongation on 3'-->5' template when fork moves in 3'-->5'
direction

The daughter DNA strand which is synthesized in discontinuous complex fashion on

the 3'-->5' template is called lagging strand. It occurs in the following steps:

i) Synthesis of Okazaki fragment:

To the RNA primer synthesized by primosome, 1000-2000 nucleotides are added by

DNA pol-III to synthesis Okazaki fragments.
ii) Excision of RNA primer:

When the Okazaki fragment synthesis was completed up to RNA primer, then RNA
primer was removed by DNA pol - I using its 5'-->3' exonuclease activity.

iii) Filling the gap (Nick translation)

The gap created by the removal of primer, is filled up by DNA pol - I using the 3'-OH
of nearby Okazaki fragment by its polymerizing activity.

iv) Joining of Okazaki fragment: (Nick sealing)

Finally, the nick existing between the fragments are sealed by DNA ligase which
catalyze the formation of phosphodiester bond between a 3'-OH at the end of one
strand and a 5' - phosphate at the other end of another fragment. The enzyme requires
NAD for during this reaction.
III) TERMINATION:

Termination occurs when the two replicating forks meet each other on the opposite
side of circular E.Coli DNA. Termination sites like A, B, C, D, E and F are found to
present in DNA. Of these sites, Ter A terminates the counter clockwise moving fork
while ter C terminates the clockwise moving forks. The other sites are backup
sites. Termination at these sites are possible because, at these sites tus protein
(Termination utilizing substance) will bound to Dna B protein and inhibits its helicase
activity. And Dna B protein released and termination result.

After the complete synthesis, two duplex DNA are found to be catenated
(knotted). This catenation removed by the action of topoisomerase. Finally, from
single parental duplex DNA, two progeny duplex DNA synthesized.
REGULATION OF PROKARYOTIC REPLICATION:

Especially initiation of replication is regulated. Dna A protein when

available in high concentration then ratio of DNA to cell mass is quiet high
but at low Dna A concentration, the ratio found to be low. This shows that
Dna A protein regulates the initiation of replication.

The sequence most commonly methylated in E.Coli is GATC including in

three of 13mer sequence. Thus, the observation that E.Coli defective in the
GATC methylation enzyme are very inefficiently replicated, suggests that
the DNA replication trigger also responds to the level of Ori C
methylation.
 EUKARYOTIC DNA REPLICATION
Eukaryotic DNA replication is not well described but the fundamental mechanism is same as
prokaryotic DNA Replication. Replications in eukaryotes are more complex. Because DNA
molecule of eukaryote
 eukaryotic genomes are quite complex,
 Considerably larger than bacterial DNA
 Organized into complex nucleoprotein structure (chromatin)
Essential features of DNA replication are the same in prokaryotes and eukaryotes, but some
variation also there.
Similarities of prokaryotes and eukaryotic replication
Replication process is fundamentally similar in both prokaryotes and eukaryotes. Process
that are similar Include
 Formation of replication fork
 Simi conservative replication
 Movement of replication fork bidirectional
 Primer synthesis
 Okazaki fragment synthesis in lagging strand
 Primer removal
 Gap bridging between newly synthesized DNA fragments.
Difference between prokaryotic and eukaryotic replication
Overall process of eukaryotic replication is bit more complex. Important differences are due
to
 Larger size of eukaryotic DNA (105-106 Kb) compared to prokaryotic DNA 15x103 kb
in E.Coli
 Distinct package of eukaryotic DNA in the term of chromatin
 Slower rate of fork movement in eukaryotes
For DNA to become available to DNA polymerase, nucleotide must dissemble. This step
slows the Rate of fork movement.
Replication rate:
Prokaryotes: An E.Coli replication fork progresses at approximately 1000 bp / sec.
Eukaryotes: Replication rate ten times slower than prokaryotes 10 bp / sec.
Enzymes and proteins required for eukaryotic DNA replication
Eukaryotic DNA polymerase:
In eukaryotes there are five different polymerases and they differ in
 Intracellular compartmentation
 Kinetic property
 Response to inhibitor

DNA pol location function

DNA Pol α nucleus lagging strand synthesis
DNA Pol β nucleus DNA repair
DNA Pol ϒ mitochondria mitochondrial replication
DNA Pol δ nucleus leading strand synthesis
DNA Pol Ɛ nucleus gap filling between okazaki fragments

DNA Pol α
- Located in nucleus
- Catalysis the synthesis of lagging strand
- Tetramer - larger subunit - 5´-3´ polymerization activity
-Two smaller subunit – primase activity
- one subunit – assist in other three subunits
- Associated with primase activity.
- Having low processivity because it is involved in lagging strand synthesis.
- RNA primer 5-15 nucleotides are subsequently extended by DNA Pol α.
DNA Pol δ
- Located in nucleus
- Catalyzes the synthesis of leading strand
- Having two subunits – larger subunits catalyzes 5´-3´ polymerization
activity
- Smaller subunits catalyzes 3´-5´ exonuclease
activity (proof reading activity)
- High processivity is due to PCNA (Proliferating cell nuclear antigen)
PCNA
- Molecular weight 25,000
- Multimeric protein
- Found in large amount in nuclei of proliferating cells.
- Act as “clamp” to keep DNA pol δ from dissociating off the leading DNA strand.
“Clamp” consist of 3 PCNA molecules each containing two topologically identical
domains that are tightly associated to form closed ring.
- DNA pol δ improves fidelity of replication by a factor of 10 2 due to its proof reading
action. It contributes in limiting the rates of overall error to 10-9 to 10-12.
- DNA Pol δ is also associated with helicase activity.

DNA Pol Є
- located in nucleus
- monomeric protein
- associated with - 5´- 3´ polymerization activity
5‟- 3‟ exonuclease activity (to remove RNA
primer) 3‟- 5‟ exonuclease activity (to proof read)
DNA pol Є catalyzes the repair mechanism and catalyzes the removal of primer and filing the
primer gap in Okazaki fragments.

Replicating factor A/ Replicating protein A (RPA/RFA)

RPA/ RFA are similar to single strand binding protein. They bind to SS DNA and prevent
the re-naturation of parental DNA.
Replication factor C (RFC)
 RFC also called as clamp loader or match maker.
 RFC assist in DNA pol δ to form clamp between DNA and PCNA.
 RFC also plays important role in setting up a link between DNA pol δ and DNA pol
α, so that the leading strand synthesis and lagging strand synthesis in eukaryotes can
take place simultaneously.

Histone Dissociation and Association

Since DNA is present in packaged form as chromatin, DNA replication is sandwiched
between two additional steps in eukaryotes.
1. Carefully ordered and in complete dissociation of the chromatin.
2. Re-association of DNA with the histone octomers to form nucleosome.
Dissociation of histone: methylation at the fifth position of cytosine residues by a DNA
methyl transferase appears to functioning by loosening up the chromatin structure. This
allows DNA access to proteins and enzymes needed for DNA replication.
Synthesis of histone: the synthesis of new histone occurs simultaneously with DNA
replication.

Sequential steps in eukaryotic DNA replication

DNA replication is a very complicated process that involves several enzymes and other
proteins. It occurs in three main stages: initiation, elongation, and termination

Initiation
ARS (Autonomously Replicating Sequences)
In eukaryotes the DNA replication is initiated at specific site known as ARS
(Autonomously Replication Sequences) or replicators.
ARS (Origin of chromosome in eukaryotes) contains
- A central core sequence which contains highly conserved 11 bp sequence (AT rich
sequence)
- Flanking sequences.
ARS – is 100- 150 long (generally it span about 150 bp)
There are multiple origins in eukaryotes. Eg: yeast contains 400 ARS. The multiple origins
are spaced 30 -300 kb apart. The sequence between two origins of replication is called
replicons. An average human chromosome contains as many as 100 replicons and
replication may proceed simultaneously at as many as 200 forks.
- The central core sequence contains 11 bp element known as “ARS consensus
sequence” rich in AT pair (It is similar to AT rich 13 mers present in E.Coli Ori C). It
is also called as ORE (Origin replication element)
- The flanking sequences consist of overlapping sequence that include varients of core
sequences

ORE (Origin Replicating Element) and ORC (Origin Recognition Complex)

 At the origin there is an association of sequence specified – ds DNA binding
sequence.
 ORE (11 bp sequence in core sequence) binds to a set of proteins (DNA pol α, DNA
pol δ, RFC, PCNA, RFA, SSB and helicase) collectively called as ORC Origin
Recognition Complex
 ORC is a multimeric protein. Initiation of replication in all eukaryotes requires this
multimeric subunit protein (ORC) which binds to several sequences within the
replicator.

DUE (DNA Unwinding Element)

ORE located adjacent to approximately 80 bp AT rich sequence that is easy to unwind. This
is called DUE (DNA Unwinding Element) Binding of ORC to ORE causes unwinding at
DUE.
Events in replication fork:
When ORC (DNA pol α, DNA pol δ, RFC, RFA, PCNA, SSB helicase into the origin of
replication especially at ORE, the DNA synthesis is initiated. The replication fork moves bi-
directionally and replication proceeds simultaneously as many as 200 forks.
Formation of replication fork:
The replication fork in eukaryotes consists of four components that form in the following
sequence.
i. DNA helicase and DNA pol δ (due to its associated helicase activity) unwinds
short segment of parental DNA at 80 bp AT rich sequence called DUE (DNA
unwinding elements) which is located adjacent to ORE.
ii. DNA pol α initiated the synthesis of RNA primer. (DNA pol α is also having
primase activity) The primer is approximately 10 bp.
 iii. DNA pol α in lagging strand and DNA pol δ in leading strand initiates the
daughter strand synthesis.
iv. SSB and RFA bind to SS DNA and prevent re-annealing of SS DNA.
In addition to the above, two additional play important role in replication of eukaryotes
i. PCNA (proliferating cell nuclear antigen) act as a „‟clamp‟‟ to keep DNA pol δ
from dissociating off the leading strand and thus increasing the processing of
DNA pol δ.
ii. RFC also called as „clamp loader‟ or „match maker‟.
RFC assist in - DNA pol δ to form clamp between DNA and PCNA and
- setting up a link between DNA pol δ and DNA pol α so that the leading
Strand and lagging strand synthesis in eukaryotes can take place simultaneously.

Rate of Replication fork Movement

The rate of replication fork movement in eukaryote (approximately 50 nucleotide /sec) is
only one tenth that observed in E.Coli at this rate, replication of an average human
chromosome proceeding from a single origin would take more than 500 hours. Instead of
that, replication of human chromosome proceeds bi-directionally from multiple origins
spaced 30-300 kb apart and completed within an hour.
DNA sequence between two origins of replication is called replicons. An average
chromosome contains nearly 100 replicons and thus replication proceeds simultaneously at as
many as 200 forks.

ELONGATION
During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3' end of
the newly synthesized polynucleotide strand. The template strand specifies which of the four
DNA nucleotides (A, T, C, or G) is added at each position along the new chain. Only the
nucleotide complementary to the template nucleotide at that position is added to the new
strand. For example, when DNA polymerase meets an adenosine nucleotide on the template
strand, it adds a thymidine to the 3' end of the newly synthesized strand, and then moves to
the next nucleotide on the template strand. This process will continue until the DNA
polymerase reaches the end of the template strand.
All newly synthesized polynucleotide strands must be initiated by a specialized RNA
polymerase called primase. Primase initiates polynucleotide synthesis and by creating a short
RNA polynucleotide strand complementary to template DNA strand. This short stretch of
RNA nucleotides is called the primer. Once RNA primer has been synthesized at the template
DNA, primase exits, and DNA polymerase extends the new strand with nucleotides
complementary to the template DNA. Eventually, the RNA nucleotides in the primer are
removed and replaced with DNA nucleotides. Once DNA replication is finished, the daughter
molecules are made entirely of continuous DNA nucleotides, with no RNA portions.

The Leading and Lagging Strands

DNA polymerase can only synthesize new strands in the 5' to 3' direction. Therefore, the two
newly synthesized strands grow in opposite directions because the template strands at each
replication fork are antiparallel. The "leading strand" is synthesized continuously toward the
replication fork as helicase unwinds the template double stranded DNA.
The "lagging strand" is synthesized in the direction away from the replication fork and away
from the DNA helicase unwinds. This lagging strand is synthesized in pieces because the
DNA polymerase can only synthesize in the 5' to 3' direction, and so it constantly encounters
the previously synthesized new strand. The pieces are called Okazaki fragments, and each
fragment begins with its own RNA primer.

Leading strand synthesis:

- Leading strand synthesis is initiated upon RNA primer, synthesized by the primase
subunit of DNA pol α. The RNA primer contains 10-15 nucleotides.
- Then DNA pol α adds a stretch of DNA to the primer.
- At this point replication factor C (RFC) carries out a process called polymerase
switching.
- RFC removes DNA pol α and assembles PCNA in the region of primer strand
terminus.
- Then DNA pol δ binds to PCNA and carries out highly processive leading strand
synthesis due to its 5‟-3‟ polymerization activity.
- After the addition of several nucleotides in the daughter strand, primer is removed.
DNA pol Є due to its 5‟-3‟ exonuclease activity removes the primer and the gap is
filled by the same DNA pol Є due to its 5‟-3‟ polymerization activity.
- Then the nick is sealed by DNA ligase.
- DNA pol δ improves the fidelity of replication due to its proof reading activity.
Lagging strand synthesis:
- Lagging strand synthesis of Okazaki fragment initiated same way as leading strand
synthesis. An Okazaki fragment contains 150-200 nucleotides.
- RNA primer is synthesised by DNA pol α due to its primase activity.
- The primer is then extended by the same DNA pol α due to its 5‟-3‟ polymerization
activity, using deoxy ribonucleotides (dNTPs).
- Priming is a frequent event in lagging strand synthesis with RNA primers placed every
50 or 80 nucleotides.
- All but one of the ribonucleotides in RNA primer is removed by RNase H1.
- Then exonuclease activity of FEN 1/ RTH 1 complex removes the one remaining
nucleotide.
- The gap is filled by DNA pol Є by its 5‟-3‟ pol activity.
- DNA ligase joins the Okazaki fragment of the growing DNA strand.

Combined activity of DNA pol α and DNA pol δ:-

Looping of lagging strand allows a combined polymerase α and polymerase δ asymmetric
dimer to assemble and elongate both leading and lagging strands in the same overall direction
of fork movement.

TERMINATION
Telomeres:
- Eukaryotic chromosomes are linear. The ends of chromosomes have specialized
structures known as „telomeres‟.
- Telomeres are – short (5-8 bp)
- tandemly repeated and
- GC rich nucleotide sequence.
- Telomeres form protective cap 7-12 kbp long in the ends of chromosome. Telomeres
are necessary for chromosome maintenance and stability. They are responsible for
maintaining chromosome integrity by protecting against DNA degradation and
rearrangement.
Problem in the completion of replication of lagging strand:
- Linear genomes including those of several viruses as well as the chromosomes of
eukaryotic cells force a special problem completion of replication of the lagging
strand.
- Excision of an RNA primer from the 5‟ end of a linear molecule would leave a gap
(primer gap). This gap cannot be filled by DNA polymerase action, because of the
absence of a primer terminus to extend. If the DNA could not be replicated, the
chromosome would shorten a bit with each round of replication.
- This problem has been solved by Telomerase.

Telomerase:
- Telomerase is ribonucleoprotein. It contains a RNA component which has repeat of
9 to 30 nucleotides long. This RNA component serves as the template for the
synthesis of telomeric repeats at the parental DNA ends.
- Telomerase is a RNA dependent DNA polymerase with a RNA component.

Telomerase uses the

- 3‟ end of parental DNA strand as primer,
- RNA component of telomerase as template,
- adds successive telomeric repeats to the parental DNA strand at its 3‟ end due to its
5‟-3‟ RNA dependent DNA polymerase activity.
Regeneration of telomeres:
Telomeric DNA consists of simple tandemly repeated sequences like those
shown as below:
Telomeric repeats sequence at 5‟end

Organism Repeat
Human AGGGTT

Higher plant AGGGTTT

Algae AGGGTTTT

protozoan GGGGTTTT
Yeast GGGT

- These sequences are repeatedly added to the 3‟ termini of chromosomal DNAs

by
„Telomerase‟. Telomerase uses its RNA component as template and parental
DNA as primer. Then by its RNA dependent DNA polymerase activity it
repeatedly adds telomeric sequences to the 3‟ termini of parental DNA.
- Then the telomerase is released.
- Finally the RNA primer, (of telomerase) is bound near the lagging strand and it is
extended by DNA polymerase. Thus the lagging strand synthesis is completed.
 CONCLUSION:

In conclusion, the comparative analysis of DNA replication in prokaryotes and

eukaryotes highlights both shared principles and distinctive features in the
replication process. While prokaryotic replication is characterized by simplicity
and efficiency due to the compact organization of genetic material in circular
chromosomes, eukaryotic replication exhibits greater complexity and regulation
to manage multiple linear chromosomes within the nucleus.

Throughout this research project, we have gained insights into the molecular
machinery involved in DNA replication, including the roles of DNA
polymerases, helicases, primases, and other accessory proteins. We have also
explored the regulatory mechanisms that ensure the fidelity and timing of DNA
replication, such as origin recognition complexes, cell cycle checkpoints, and
chromatin remodeling factors.

Furthermore, by examining the evolutionary implications of the differences in

DNA replication between prokaryotes and eukaryotes, we have gained a deeper
understanding of how genomic organization and complexity have evolved over
time. The variations in replication machinery and regulatory strategies reflect
the diverse evolutionary pressures faced by different organisms and contribute
to their adaptability and survival in changing environments.

Overall, this research project underscores the importance of DNA replication as

a fundamental biological process and provides a foundation for further
exploration into the molecular mechanisms and evolutionary dynamics of
genome duplication. By continuing to unravel the intricacies of DNA
replication in prokaryotes and eukaryotes, we can unlock new insights into
cellular biology, genetics, and evolutionary biology, with potential applications
in medicine, agriculture, and biotechnology.
Through collaborative efforts and interdisciplinary research, we can continue to
deepen our understanding of DNA replication and its significance in shaping the
diversity and complexity of life on Earth.
 SUMMARY:

The study of DNA replication in prokaryotes and eukaryotes provides crucial

insights into the fundamental processes governing genetic inheritance and
cellular function. In prokaryotes, DNA replication is relatively simple,
occurring in circular chromosomes, while in eukaryotes, it is more complex due
to the presence of multiple linear chromosomes within the nucleus.

Key similarities exist between prokaryotic and eukaryotic DNA replication,

including the involvement of DNA polymerases, helicases, primases, and DNA
ligases in synthesizing new DNA strands. However, there are also significant
differences, such as the presence of multiple DNA polymerases and additional
regulatory factors in eukaryotic replication.

Regulatory mechanisms play a vital role in ensuring the fidelity and timing of
DNA replication in both prokaryotes and eukaryotes. These mechanisms
include origin recognition complexes, cell cycle checkpoints, and chromatin
remodeling proteins, which coordinate the replication process and maintain
genome stability.

The evolutionary implications of the differences in DNA replication between

prokaryotes and eukaryotes highlight the adaptive strategies employed by
different organisms to cope with diverse environmental challenges.
Understanding these evolutionary dynamics provides valuable insights into the
origins and diversification of life on Earth.

Overall, the study of DNA replication in prokaryotes and eukaryotes contributes

to our understanding of fundamental biological processes and has broad
implications for fields such as genetics, molecular biology, and evolutionary
biology. By continuing to investigate the molecular mechanisms and
evolutionary dynamics of DNA replication, we can further unravel the
complexities of life and develop innovative solutions to biological challenges.
 Bibliography:

1. Alberts, B., Johnson, A., Lewis, J., et al. (2002). Molecular Biology of the
Cell. 4th edition. New York: Garland Science.

2. Bell, S. P., & Dutta, A. (2002). DNA replication in eukaryotic cells.

Annual Review of Biochemistry, 71, 333-374.

3. Kornberg, A., & Baker, T. (1992). DNA Replication. 2nd edition. New
York: W. H. Freeman.

4. O'Donnell, M., Langston, L., & Stillman, B. (2013). Principles and

concepts of DNA replication in bacteria, archaea, and eukarya. Cold
Spring Harbor Perspectives in Biology, 5(7), a010108.

5. Watson, J. D., & Crick, F. H. (1953). Molecular structure of nucleic

acids: A structure for deoxyribose nucleic acid. Nature, 171(4356), 737-
738.

6. DePamphilis, M. L. (1999). DNA replication in eukaryotic cells. Cold

Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

7. Reyes-Lamothe, R., Nicolas, E., & Sherratt, D. J. (2012). Chromosome

replication and segregation in bacteria. Annual Review of Genetics, 46,
121-143.