DNA Replication

INTRODUCTION

Transfer of genetic information from parent to progeny

Complex process
Takes place during interphase between 2 mitotic cycles, more
specifically during S phase.
Not completely understood
DNA is autocatalytic and heterocatalytic

Watson and Crick DNA model implies a mechanism for

replication:
a. Unwind the DNA molecule.
b. Separate the two strands.
c. Make a complementary copy for each strand.
 Basic rule for replication

Nucleotide monomers are added one by one to the end of a

growing strand by DNA Polymerase enzyme

Sequences in daughter strand is complimentary to the parent

strand

HOW THE 2 STRANDS OF A DAUGHTER MOLECULE ARE

RELATED TO THE 2 STRANDS OF PARENT MOLECULE???
 Delbruk suggested 3 modes of DNA replication

1. Conservative ( 1 entirely new and 1 entirely old)

2. Semi conservative ( one strand new one strand old in

same helix)

3. Dispersive ( patch work of old and new)

Mode of replication
The Meselson - Stahl experiment
 Origin of replication

Site where replication begins

1 in E. coli
1,000s in human

Strands are separated to allow replication machinery contact

with the DNA
Many A-T base pairs, because easier to break 2 H-bonds
than 3 H-bonds
 Direction of replication

Unidirectional (eg:- replication of mt DNA in vertibrates,

prokaryotic DNA)

Bidirectional (eg:- eukaryotic DNA)

 Enzymes of DNA replication

Nuclease
hydrolyse the phosphodiester bonds
exonuclease
endonuclease

Polymerase or replicase
catalyse the formation of polymers
 Prokaryotic DNA replication
RAW MATERIALS
ENZYMES

TEMPLATES

RNA PRIMERS

REPLICONS

REPLISOMES

PRIMOSOMES

oENZYMES
DNA polymerase (I, II, III)

DNA helicases/ DnaB protein

Topoisomerase/ gyrase

SSBP
 DNA polymerase I

Discovered byArthur Kornbergin 1956,it was the first knownDNA

polymerase
Composed of 928 aa
Single 102KD polypeptide
5 3 endonuclease activity, nick translation during DNA repair
3 5 exonuclease activity, Proof reading

Functions:
1. Template site for binding template DNA
2. Primer site to bind primer RNA
3. Used to fill gap between Okazaki fragments that are formed during lagging
strand synthesis.
4. Catalyse DNA repair and discontinuous DNA synthesis.
5. Dna Repair.
 DNA polymerase II
90KD Polypeptide
Coded by PolB gene
Functions:
1. Assist in polymerisation
2. Mainly involved in DNA repair
DNA polymerase III
. synthesizes base pairs at a rate of around 1000 nucleotides per
second
. Complex molecule
. Act as 5 3 and 3 5 exonuclease
. Formed of 10 subunits
Functions:
1. Chain elongation in leading strand
2. Essential for in vivo DNA replication
3. Help in repair
 DNA helicase/ Dna B protein
Involved in strand separation
ATP dependent enzyme
2 types
> Pri A protein
> Rep protein
Pri A protein
or helicase II and III
Moves on 5 3
Attaches to the template for the lagging strand
Rep protein
Direct leading strand synthesis
Moves on 3 5
In rolling circle replication
 Topoisomerase or gyrase
Causes topological changes in DNA
Based on whether they cause single strand or double strand
break it is of 2 types

a> Type I topoisomerase

Topo I and Topo III
Temporary single strand breaks
Relaxes negative supercoils

b> TypeII topoisomerase

Topo II and Topo IV
Break and reseal both strands
 SSBP
Single strand binding protein
Tetramer
Product of SSB gene
No sequence specificity
Helps in unwinding and prevent rewinding

RNA PRIMER
Short oligonucleotide to start replication
Produced with the help of DNA primase/RNA polymerase
Hydrogen bonded to DNA
o REPLICONS

A discrete unit which helps in DNA replication

In E.coli 1 replicon is present (OriC)
o REPLISOMES

Carry out leading and lagging strand synthesis in a coordinated

manner
complex molecular machine that carries outreplicationof DNA.
o PRIMOSOMES
proteincomplex responsible for creatingRNAprimerson single
strandedDNAduringDNA replication
Consists of primase molecule linked to DNA helicase
Moves along with replication fork and synthesis RNA primer
DnaGprimase,DnaB helicase,DnaChelicase assistant,DnaT,PriA,Pri B,
andPriC
Prokaryotic DNA replication
 1. Initiation
DNA at the origin of replication denatures to expose the bases
creating a replication fork.
bidirectional
one origin, oriC, which has:
a. A minimal sequence of about 245 bp required for initiation.
b. Three copies of a 13-bp AT-rich sequence.
c. Four copies of a 9-bp sequence.
Events
a. Initiator proteins DnaA attach.
b. DNA helicase (from dnaB) binds initiator proteins on the DNA, and
denatures the AT-rich region using ATP as an energy source.
c. DNA primase (from dnaG) binds helicase to form a primosome, which
synthesizes a short (510nt) RNA primer. .
 2. Elongation

Requires
DnaB - Unwinding
primase - primer addition
DNA pol III - elongation
SSBP - prevent rewinding
RNAse H - removes RNA primer
DNA pol I - fill the gap
DNA ligase - join the okazaki fragments
Discontinous synthesis of lagging
strand
Multiple primer needed
RNA primer synthesized by primase
Okazaki fragments are formed
DNA pol I removes primer and adds
nucleotides
DNA ligase form phosphodiester
bonds that link free 3 end of
primer replacement of 5 end of
okazaki fragment.
Continuous synthesis of leading
strand
Need only one primer
RNA primer made by RNA pol
No okazaki fragments are formed
DNA pol III for elongation in
5--3
DNA pol I removes primer and
adds nucleotides
DNA ligase gives the final touch
up
 3. Termination

Occur at Ter site (7 identical non palindromic 23bp :Ter A, Ter

D, Ter E, Ter F and Ter G )
Both clockwise( Ter G) and anti clock wise (Ter E)

Bidirectional replication of circular DNA molecules

Some circular chromosomes (e.g., E. coli) are circular throughout

replication, creating a theta-like () shape. As the strands
separate on one side of the circle, positive supercoils form
elsewhere in the molecule.
Topoisomerases relieve the supercoils, allowing the DNA strands
to continue separating as the replication forks advance
Bidirectional replication of circular DNA molecules
 Theta Replication

Circular DNA in bacteria

Replication bubble formed

from DNA unwinding and
strands separation

Replication fork point

where two strands separate

Continues bi-directionally
until they meet
 Rolling Circle Replication
begins with a nick (single-stranded break) at the origin
The 5 end is displaced from the strand
3 end acts as a primer for DNA polymerase III, which
synthesizes a continuous strand
The 5 end continues to be displaced as the circle rolls, and is
protected by SSBs until discontinuous DNA synthesis makes it a
dsDNA again
During viral assembly it is cut into individual viral chromosomes
and packaged into phage head.
 Eukaryotic DNA replication
not as well understood as bacterial replication
more complex
Large linear chromosomes

Tight packaging within nucleosomes

More complicated cell cycle regulation

In 1968, Huberman and Riggs provided evidence for the multiple
origins of replication
DNA replication proceeds bidirectionally from many origins of
replication
ENZYMES
 DNA pol a is the only polymerase to associate with primase
The DNA pol a/primase complex synthesizes a short RNA-
DNA hybrid

10 RNA nucleotides followed by 20 to 30 DNA
nucleotides
This is used by DNA pol d or e for the processive elongation
of the leading and lagging strands
Current evidence suggests a greater role for DNA pol d

The exchange of DNA pol a for d or e is called a polymerase

switch
It occurs only after the RNA-DNA hybrid is made
 DNA polymerases also play a role in DNA repair
DNA pol b is not involved in DNA replication
It plays a role in base-excision repair

Removal of incorrect bases from damaged DNA
Recently, more DNA polymerases have been identified
Lesion-replicating polymerases

Involved in the replication of damaged DNA

They can synthesize a complementary strand over the abnormal
region
 Helicase
4 types
Helicase A
Helicase
Helicase
RF-A

Topoisomerase
2 types
Class I ( Topo I & Topo III)

Class II ( Topo II & Topo IV)

SSBP
Human SSBP or RP-A

Tetramer
 Origins of Replication
origins of replication found in eukaryotes have some similarities
to those of bacteria

They are 100-150 bp in length

They have a high percentage of A and T

They have three or four copies of a specific sequence
Similar to the bacterial DnaA boxes
Origin recognition complex (ORC)
A six-subunit complex that acts as the initiator of eukaryotic
DNA replication

It appears to be found in all eukaryotes
Requires ATP to bind ARS elements
Single-stranded DNA stimulates ORC to hydrolyze ATP
 Initiation
Multiple origin
Histones associated with DNA should be removed
Rate 105 bp/min
Takes 1000 times more replication time than that of prokaryotic
replication
Large amount of DNA in chromosome at multiple replisomes
Initiative protein selects the origin and activates it with the help of
other proteins
Initiatve protein is ORC
Y shaped intermediate will form
 Elongation
Primer excised by endonuclease or RNaseH

Leading and lagging strand synthesis by the coupled action of Polymerase

and Helicase
Histone reassociation after synthesis

Nucleosome assembly

Termination
Occurs by telomere replication
Telomeric sequences consist of
Moderately repetitive tandem arrays
3 overhang that is 12-16 nucleotides long
Telomeric sequences typically consist of
Several guanine nucleotides
Often many thymine nucleotides
 DNA polymerases possess two unusual features
1. They synthesize DNA only in the 5 to 3 direction
2. They cannot initiate DNA synthesis
These two features pose a problem at the 3 end of linear chromosomes
 Therefore if this problem is not solved
The linear chromosome becomes progressively shorter with
each round of DNA replication
Indeed, the cell solves this problem by adding DNA sequences to
the ends of telomeres
This requires a specialized mechanism catalyzed by the enzyme
telomerase
Telomerase contains protein and RNA
The RNA is complementary to the DNA sequence found in
the telomeric repeat

This allows the telomerase to bind to the 3 overhang
 Step 1 = Binding

The binding-
polymerization- Step 2 = Polymerization
translocation cycle can
occurs many times

This greatly lengthens

one of the strands
Step 3 = Translocation

The complementary
strand is made by primase,
DNA polymerase and ligase

RNA primer
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