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REPLICATION



M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

Watson and
crick
Introduction
Besides maintaining the integrity of DNA sequences
by DNA repair, all organisms must duplicate their
DNA accurately before every cell division.
DNA replication occurs at polymerization rates of
about 500 nucleotides per second in bacteria and
about 50 nucleotides per second in mammals.
Clearly, the proteins that catalyze this process must
be both accurate and fast.
Speed and accuracy are achieved by means of a
multienzyme complex that guides the process and
constitutes an elaborate "replication machine."
Replication occurs in 5

to 3

direction only.

Replication is simultaneous on both strands.
Replication is bidirectional.
Replication obeys base pair rule

Replication results in 2 daughter DNA strands.
Each daughter DNA strand has one
parent strand and one complementary
strand synthesized newly. Hence this
Replication is semi-conservative.
Held by phospho-di-ester bonds
and Hydrogen bonds


CELL - CYCLE
Cell cycle is a sequence of events that occur in
a cell during cell division.
It results in formation of 2 identical daughter
cells.
Duration of cell cycle varies from cell to cell.
It occurs in 4 phases
G
1
PHASE [ gap-1]
S PHASE [synthetic]
G
2
PHASE [gap-2]
M PHASE [ mitotic]
G
0

CELL- CYCLE
G
1
phase ; Preparative phase for DNA synthesis. All cellular
components replicate except DNA . Cell size
increases. Any damage to DNA is detected.

S phase ; DNA replication takes place.

G
2
phase ; Prepares for cell division and spindle
formation. Any damage to DNA is detected.

M phase; Cell undergoes cell division . It includes prophase
,metaphase, anaphase ,and telophase.

After mitosis cell may continue cycle by re-entering into G
1
or
enter G
0
and remain dormant or leads to cell
death

MODELS FOR DNA REPLICATION
These are many hypothesis to explain
the process of replication. They are
1. Conservative model
2. Semi conservative model
3. Dispersive model
SEMI CONSERVATIVE MODEL OF REPLICATION
REPLICATION IS SEMICONSERVATIVE

SEMI CONSERVATIVE MODEL OF REPLICATION

DNA-REPLICATION
Requirements
1.Deoxyribonucleotides [ dATP, dGTP, dCTP, dTTP ]
2.Template DNA strand [parent strand]
3.RNA primer
4.Enzymes DNA polymerase
Primase
Helicase
DNA Ligase
Topo-isomerases
Single Strand Binding Proteins.

Single strand binding protein (SSBP )

Binds to ssDNA
Has two function
1. prevents reannealing , thus providing ss
template
required by polymerases
2. protects ssDNA from nuclease activity

Show cooperative binding
Helicases
Separate the ds DNA to ss DNA by dissolving the
hydrogen bonds holding the two strands together
These separates dsDNA at physiological temperature
ATP dependent
At least 9 helicases have been described in E coli
Of which DNA binding protein A, B , C ( Dna A,
Dna B, Dna C ) are most important
Initial separation is by Dna A
Continued further by Dna B ( major strand
separating protein acts bidirectionally )
Dna C is required for loading Dna B at site of
replication


PRIMASE:
Primase is a specilised RNA polymerase
It synthesis a short strech of RNA in 5 3
direction on a template running in 3 5
direction.
An RNA primer, about 100-200 nucleotides
long, is synthesized by the RNA primase.
The RNA primer is removed by DANP, using
exonuclease activity and is replaced with
deoxyribo nucleotides by DNAP
DNA Ligases
DNA ligases close nicks in the phosphodiester backbone
of DNA. Two of the most important biologically roles of
DNA ligases are:
1. Joining of Okazaki fragments during replication.
2. Completing short-patch DNA synthesis occurring in
DNA repair process.
There are two classes of DNA ligases:
1. The first uses NAD
+
as a cofactor and only found in
bacteria.
2. The second uses ATP as a cofactor and found in
eukaryotes, viruses and bacteriophages.
DNA LIGASE STRUCTURE

DNA Ligase Mechanism
The reaction occurs in three stages in all DNA
ligases:
1.Formation of a covalent enzyme-AMP
intermediate linked to a lysine side-chain in the
enzyme.
2.Transfer of the AMP nucleotide to the 5-
phosphate of the nicked DNA strand.
3.Attack on the AMP-DNA bond by the 3-OH of
the nicked DNA sealing the phosphate
backbone and resealing AMP.

As two strands unwind ,they result in the formation
of positive supercoils ( super twists ) in the region
of DNA ahead of replication fork.

Accumulation of these supercoils interfere with
further unwinding of ds DNA.

This problem is solved by the enzyme
Topoisomerases.

These catalyze the interconvertion of topoisomers of
DNA

Catalyze in a three step process
1. cleavage of one or both strands of DNA
2. passage of a segment of DNA through
this break
3. resealing of the DNA

Two types of topoisomerases are present
DNA which different in the linking numer
Linking number = (Twist +Wreth) 3 dimentional
-type I topoisomerases
-type II topoisomerases
Topoisomerases I
Reversibly cut one strand of double helix

Have both nuclease ( strand cutting ) & ligase (
strand resealing )

Donot require ATP ,rather use the energy released
by phosphodiester bond cleavage to reseal the nick

Removes only negative super coils
Ex : bacteria


Topoisomerases II ( DNA gyrase )

Heterodimer with 2 swivelase & 2 ATPase subunits

Swivelase subunit catalyzes trans esterification reaction
that breaks & reforms the phosphodiester backbone

ATPase subunit hydrolyzes ATP to trigger
conformational changes that allow a double helix to pass
through the transient gap
Possitive super coiled

DNA POLYMERASES
These are the enzymes responsible for the
polymerisation of deoxy ribo nucleosides,
triphosphates on a DNA template strand to
form a new complementary DNA strand.
In prokaryotes based on site and
conditions of action. They are divided into
3 types: I II III.


Common properties:
1. All polymerases can synthesis a new strand
of DNA in 5 to 3 direction. On a template
strand which is running in 3to 5 direction.
2. They also show Exo nuclease activity ( it
cleaves the end terminals of DNA) in 3to 5
direction.
3. All DNA polymerases cannot initiate the
process of replication on their own. This is
the basic defect of DNAP synthesis of new
strand .

COMPARISON OF PROKARYOTIC &
EUKARYOTIC DNA POLYMERASE
Prokaryotic Eukaryotic
FUNCTION
l Gap filling &synthesis
of lagging strand
ll DNA proofreading &
repair
DNA repair
gamma Mitochondrial DNA
synthesis
lll leading strand
synthesis

REPLICATION
There are three phases of replication
1. Initiation
2. Elongation
3. Termination
STEPS IN DNA-REPLICATION
1.Recognition of origin of replication and Un-
winding of double stranded DNA

2.Formation of replication bubbles with 2
replication forks for each replication bubble.

3.Initiation and elongation of DNA strand.

4.Termination and Reconstitution of chromatin
structure.
UNWINDING OF DS DNA

INITIATION OF DNA-REPLICATION
1.Identification of the origins of replication.
The origin of replication [oriC locus] rich in
AT pairs is identified.
A specific protein [Dna A] binds to the oriC and
results in unwinding of ds DNA.
Un winding of DNA results in formation of
replication bubble with 2 replication forks.
Ss binding proteins binds to DNA to each strand
to prevent re-annealing of DNA.
Helicases continues the process of un winding.
Topoisomerases relieve the super coils formed
during unwinding.



Topo-isomerases
DNA-REPLICATION
2.Fomation of replication fork
replication fork has 4 components
1.helicase [unwinds ds DNA]
2.primase [synthesizes RNA primer]
3.DNApolymerase[synthesizes DNA]
4.ss binding proteins [stabilizes the strand]


2.ELONGATION OF DNA
Requires RNA primer, DNA template , DNAP enzyme
and deoxyribonucleotides [dATP,dGTP ,dCTP, dTTP]

DNA polymerase catalyze the stepwise addition of
deoxyribonucleotides to 3
1
end of template strand and
thus copies the information from the template DNA.

DNAP requires RNA primer to start elongation.

DNAP copies the information from DNA template


2.ELONGATION OF DNA
1.continous synthesis occurs towards the
replication fork [leading strand] by DNA
polymerase.

2.discontinuous synthesis occurs away
from the replication fork in pieces called as
okazaki fragments which are ligated by DNA
ligase [lagging strand]. It requires multiple
RNAprimers.


Okazaki fragments

First demonstrated by Reiji Okazaki

Short fragments of DNA present on the lagging strand
resulted by retrograde synthesis.

Okazaki fragments in human cells average about 130 -
200 nucleotide in length

In E coli they are about ten times this.

REPLICATION
RNA primer is removed by DNAP with
exonuclease activity. Again the gap is filled by
DNAP. The two Okazaki pieces are later joined by
DNA ligase.
ROLE OF TELOMERS IN EUKARYOTIC
REPLICATION
A small portion of 3
1
end of parent strand is
not replicated and length of chromosome
reduces.
Telomeres play a crucial role in eukaryotic
replication.
Telomeres contain the repeat sequence of
[TTAGGG]
n .
They prevent the shorting of chromosome
with each cell division by an enzyme
telomerase.
Telomerase enzyme synthesizes and
maintains the telomeric DNA.
Telomerase adds repeats to 3
1
end of DNA
3.TERMINATION OF DNA REPLICATION
In prokaryotes the process of replication is
terminated when the two replication forks
moving in opposite directions from the origin
meet.

In E.coli replication of circular DNA takes
about 30 minutes.

In eukaryotes replication is terminated when
entire DNA is duplicated in S phase of cell
cycle.
INHIBITORS OF REPLICATION
1.Inhibitors of DNA; Prevents un-winding of DNA.
E.g. actinomycin, mitomycin

2.Inhibitors of deoxy-ribonucleotides;
E.g. Anti-folates [ inhibits Purine\
Pyrimidine synthesis]

3.Inhibitors of replicative enzymes;
E.g. norflox [inhibit DNA gyrase]
ciploflox



Replication in Eukaryotic cells
More complex than prokaryotic replication

Semicoservative ,occurs bidirectional from many oigins forming multiple
replication bubbles
Eg:- replication of Drosophilia chromosomes
single Ori C ---16 days to replicate
multiple Ori C ---3 min ( 6000 replication forks )

Sequence functionally similar to Ori C have been identified in yeast & are
called ARS ( autonomously replicating sequence )

ARS span about 300bp ( conserved sequence )

There are about 400 ARS elements in yeast

Eukaryotic DNA polymerases

Type Location Major role
Nucleus Replication of nuclear DNA
Gap filling & synthesis of lagging
strand
Nucleus Proof reading & Repair of nuclear
DNA
Mitochondria
l
Replication of mitochondrial DNA
Nucleus Replication of nuclear DNA
Leading strand synthesis

Nucleus Repair of nuclear DNA
Replication in linear genome

Problem arise with replication of ends of linear genome
( Telomers )

Removal of RNA primer on the lagging strand produces a daughter DNA with
an incomplete 5 end

If not synthesized shorter and shorter daughter DNA would result from
successive rounds of replication

This problem is solved by the enzyme TELOMERASE



Telomers
Ends of the eukaryotic linear chromosomes

Contains thousands of hexameric repeats ( TTAGGG )

Some shortening of this telomer is not a problem as they donot encode for
proteins

Cell is no longer able to divide & is said tobe senescent if shortening occurs
beyond some critical length

In germ cells ,stem cells as well as in cancer cells ,telomers donot shorten &
the cells do not senesce.( due to the presence of
Telomerase enzyme )
Telomerase

Ribonucleoprotein enzyme ( reverse transcriptase )
catalyzing the elongation of the 3 ending strand

Contains a RNA molecule that serves as the template
for the elongation of the telomeric end

Highly processive hundreds of nucleotides are added
before it dissociates
THANK YOU