DNA replication in eukaryotes

DNA replication In Eukaryotes and Role of Enzymes

1. 1

Definition
DNA replication is fundamental part of life cycle.The process by which DNA of a living
organism gives rise to a copy of itself is called DNA replication.A single DNA molecule
is copied to produce two ide ntical DNA molecules.Replication is part of
reproduction,development and maintaining normal life.Successful duplication of genetic
material decide the life and death of an organism.Replication takes place in S-phase of
cell cycle.It is necessary process because through this process genetic information
present in cells can be passed on to the daughter cells following cell division.
Models of DNA Replication
Delbruck suggested that Watson and Crick model of DNA can be replicated by three
ways. These are following as under,
 Semi-conservative Replication
 Conservative Replication
 Disreptive Replication
Semi-conservative Replication
During DNA replication a double stranded DNA is separated and each strand is used as
template for the synthesis of new strand .This results in the formation of two
copies.Each contains one original strand and one new daughter strand.
Conservative Replication
The conservative model stated that parental double helix would remain intact and
generate DNA copies consisting of entirely new molecules.
Disreptive Replication
The disreptive model predicted that parental DNA would dispersed and new copy of
DNA contains mixture of new and old DNA strands.

2. 2


Correct Model
In 1958 Mathew Meselson and Franklin Stahl of California Institute of Technology
proved that DNA replicates according to semiconservative model of replication.
Requirement for the DNA replication
 Template DNA
 Nucleotides
 Replisomes
 Replicons
 MCM Helicase
 Topoisomerase
 Primase
 DNA polymerases

3. 3

Template DNA
Each strand of DNA double helix can act as template for the synthesis of new
complementary strand.
Nucleotides
DNA molecule is composed of thousand of nucleotides that are made up of nitrogenous
bases, deoxyribose sugar and phosphate group.
Replisomes
Genomic replication requires a large collection of proteins properly assembled at DNA
replication fork and this replication require multiple steps of enzymetic pathway at
leading and lagging strandthrough replisome.

Replicons
DNA replication in eukaryotes is initiated at many points, called origin of replication or
replicons and it occur in both direction.These multiple replicons was first identified in
Saccharomyces cerevisia in 1979.

4. 4

MCM Helicase
MCM stands for minichromosome maintenance.MCM was first identified in
S.cerevisia.The MCM 2-7 complexes serve as eukaryotic replicative helicase.It is
molecular motor that hydrolyzes the ATP and unwinds the DNA at origin of replication.
Topoisomerase
It is an isomerase enzyme that prevent from supercoiling of DNA during replication.In
prokaryotes it prevent from negative supercoils only but in case of eukaryotes it prevent
from both negative and positive supercoils.
Primase
Primase is an enzyme that synthesizes short RNA sequences called primers.These
primers serve as starting point for DNA synthesis.
DNA Polymerase
In eukaryotic DNA, there are several DNA polymerases that are involved in replication
are following as under,
 Polymerase Alpha
 Polymerase Beta
 Polymerase Delta
 Polymerase Epsilon
 Polymerase Gamma
Polymerase Alpha
It starts replication at primer,repairs mistakes during proof reading.It doesn’t replicate
DNA at restricted temperature.It is associated with primase therefore sometimes known
as alpha primase.
Polymerase Beta
It recombines chromosomes during proof reading.
Polymerase Delta
It synthesizes lagging strand and is also involved in 3 to 5 prime exonucleases activity.It
is associated with protein known as proliferating cell nuclear antigen PCNA.In the
presence of PCNA, polymerase delta is highly active.

5. 5

Polymerase Epsilon
It synthesizes leading strand.It is involved in 3 to 5 prime exonucleases activity.It has
organizational role and associated with clamp proteins or PCNA ring for the replication.
Polymerase Gamma
It replicates mitichondrial DNA.
Mechanism of DNA Replication
DNA unwinding and DNA synthesis are assembled in multistep process.There are
three major steps that are invovled in DNA replication.
 Initiation
 Elongation
 Termination
Initiation
Initiation of DNA replication is the first stage of DNA synthesis and it starts from specific
sequences called origin of replication and eukaryotic cells have multiple origin of
replication.
Pre-replication Complex
Pre-replication complex forms at origin of replication during G1 phase.In most
eukaryotes it is composed of ORC ,Cdc6,Cdt1 and MCM. ORC stands for origin
replication complex .It recognizes and binds with origin of replication.All the other
proteins Cdc6,Cdt1 and MCM bind with ORC and this whole complex is known as pre-
initiaion complex. First of all Cdc6 known as cell division cycle 6 binds with ORC.After
that Cdt1 which is known as chromatin liscensing DNA replication factor 1 helps
minichromosome maintenance protein complex to bind with ORC.
Initiation Complex
The main purpose of Cdc6 and Cdt1 is to load MCM on DNA.After the loading of MCM
these proteins are phosphorylated for the activation of initiation complex.This complex is
formed during the transition of G1 phase to S phase.Cyclic dependent kinase CDK and
Dbf4 protein kinase phosphorylate Cdc6 and Cdt1 and make this complex active for the
unwinding of DNA.

6. 6

Activator Complex
After the activation of initiation complex two more complex factors Cdc45 and GIN
activates the MCM helicase activity.Therefore this complex is known as activator
complex which is formed during the Sphase of cell cycle.It unwinds the DNA and
replication fork is formed.

Elongation
Replication Fork
In humans,synthesis of new DNA occurs at the rate of 300 nucleotides per minute.Once
initiation complex is formed the cell is passed into S Phase andreplication fork is
formed .This complex then becomes a replisome.The eukaryotic replisome complex is
responsible for coordinating of DNA replication.It consists of polymerase enzymes
,clamp loader proteins, Topoisomerase and all other important factors that help in
replication of DNA strands.
Replication Protein A (RPA)
RPA is major protein that binds to single stranded DNA in eukaryotic cells.RPA
prevents DNA from winding back on itself or forming secondary structures.It keeps the
DNA unwound for polymerase to replicate it.

7. 7


Leading Strand
Leading strand is template strand and it is replicated in the same direction as the
movement of replication fork.Replication is continuous along the leading strand.It
occurs from 5' to 3' direction.Many polymerase enzymes and proteins are invovled in
the replication of leading strands.
Mechanism
After the unwinding of DNA through MCM helicase polymerase epsilon associated with
PCNA ring then starts replication. Polymerase epsilon also involve in exo-nucleases
activity but there are minimum chances of error during the replication of leading
strand.Therefore replication along the leading strand is continuous.
Lagging strand
DNA replication on the lagging strand is discontinuous and it is away from the
replication fork. Although newly formed DNA strand also run in 5' to 3' direction.
Mechanism
In lagging strand synthesis, DNA polymerase will synthesize short fragments of DNA
called Okazaki fragments which are added to the 3' end of the primer. These fragments
can be anywhere between 100-400 nucleotides long in eukaryotes.Polymerase alpha
and primer initiate replication on lagging strand.Then PCNA dependent DNA
polymeraseδ starts adding nucleotides.When polymerase δcomes in direct contact with
upcoming primer.It starts cleaving 2-3 RNA sequences from primer region,because after
the replication there is need to remove RNA primer.This removal needs another protein
known as Fen1 Protein.It works with polymeraseδ. It starts cleaving the rest of RNA
primer sequences. After the removal of nucleotide sequences the gaps are left which
are filled by polymerase δ.Once it will fill the gaps it creates nick.

8. 8

Nick is the discontinuity between DNA fragments due to the enzyme action and removal
of phosphodiesterbond.Nick is then filled with the help of ligase I.This process is known
as Okazaki fragment maturation.

Termination
In eukaryotes DNA polymerase cannot replicate the terminal DNA segment of lagging
strand of linear chromosomes. The terminal region of DNA is known as
telomer.Telomers must have unique features and enzymes that facilitates replication.
Mechanism
Terminal region of linear DNA which is known as telomere becomes shorter and
shorter.This is known as end replication problem.An enzyme known as telomerase is
used to solve this problem.Telomerase is RNA containing enzyme.This was discovered
in 1958 by Elizabeth Blackburn and Caral Greider.Telomerase can reverse transcribe
onto its own RNA template to synthesize additional DNA.Now 3' telomere is longer.

9. 9

Comparisons Between Prokaryotic and Eukaryotic DNA
Replication



Prokaryotic DNA Replication



Eukaryotic DNA replication
Occurs inside the cytoplasm. Occurs inside the nucleus.
Replication occurs at only one point of
DNA molecule.
Replication occurs at several points.
Origin of replication is formed of about
100-200 nucleotides.
Origin of replication is formed of about 150
nucleotides.
Initiation is carried by DnaA and DnaB.
Initiation is carried out by several complex
proteins and ORCs.
Only one replication fork is formed.
Multiple replication forks are formed
simultaneously in each chromosome.
There is one origin of replication. Has multiple replicons.
Okazaki fragments are 1000-2000
nucleotides long.
Okazaki fragments are short contain 100-
200 nucleotides.
DNA gyrase is needed. DNA gyrase is needed.
Replication is very rapid. Replication is very slow.

10. 10

References
Molecular Cell Biology By Harvey Lodish
Molecular Biology of cell By Bruce Alberts
Molecular and Cell Biology for Dummies By Rene Fester
Molecular and Cell bio by Horst Feldmann
Genetics and Molecular Biology by Robert Schleif
Campbell biology by Jane B. Reece, Michael L.Chain, Steven A.Wassermen ,Robert
B.Jackson
Principle of Genetics by D. Peter Snustad and Michael J. Simmons
http://study.com/academy/lesson/what-is-dna-replication-conservative-semi-
conservative-dispersive-models.html
https://www.ebi.ac.uk/interpro/potm/2006_1/Page1.htm
https://www.ncbi.nlm.nih.gov/pubmed/12806123
http://www.nature.com/scitable/definition/rna-polymerase-106
https://www.boundless.com/microbiology/textbooks/boundless-microbiology-
textbook/microbial-genetics-7/dna-replication-73/dna-replication-in-eukaryotes-421-
12941/