Besides maintaining the integrity of DNA
sequences by DNA repair, all organisms must
duplicate their DNA accurately before every cell
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
• 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
Duration of cell cycle varies from cell to cell.
It occurs in 4 phases
G1 PHASE [ gap-1]
S PHASE [synthetic]
G2 PHASE [gap-2]
M PHASE [ mitotic]
G1 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.
G2 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 G1
or enter G0 and remain dormant or leads to cell
MODELS FOR DNA REPLICATION
These are many hypothesis to
explain the process of replication.
1. Conservative model
2. Semi conservative model
3. Dispersive model
1.Deoxyribonucleotides [ dATP, dGTP, dCTP, dTTP ]
2.Template DNA strand [parent strand]
4.Enzymes DNA polymerase
Single Strand Binding Proteins.
Single strand binding protein (SSBP )
Binds to ssDNA
Has two function
1. prevents reannealing , thus providing ss
required by polymerases
2. protects ssDNA from nuclease activity
Show cooperative binding
Separate the ds DNA to ss DNA by dissolving the
hydrogen bonds holding the two strands together
These separates dsDNA at physiological temperature
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
Primase is a specilised RNA polymerase
It synthesis a short strech of RNA in 5’ 3’
direction on a template running in 3’ 5’
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 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
2. The second uses ATP as a cofactor and found in
eukaryotes, viruses and bacteriophages.
DNA Ligase Mechanism
The reaction occurs in three stages in all
1.Formation of a covalent enzyme-AMP
intermediate linked to a lysine side-chain in
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
These catalyze the interconvertion of topoisomers of
Catalyze in a three step process
1. cleavage of one or both strands of DNA
2. passage of a segment of DNA through
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
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
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.
1. All polymerases can synthesis a new
strand of DNA in 5’ to 3” direction. On a
template strand which is running in 3’to
2. They also show Exo nuclease activity ( it
cleaves the end terminals of DNA) in 3’to
3. All DNA polymerases cannot initiate the
process of replication on their own. This is
the basic defect of DNAP synthesis of new
COMPARISON OF PROKARYOTIC &
EUKARYOTIC DNA POLYMERASE
Prokaryotic Eukaryotic FUNCTION
l α Gap filling &synthesis
of lagging strand
ll ε DNA proofreading &
β DNA repair
gamma Mitochondrial DNA
lll δ leading strand
There are three phases of replication
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
3.Initiation and elongation of DNA strand.
4.Termination and Reconstitution of
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
2.Fomation of replication fork
replication fork has 4 components
1.helicase [unwinds ds DNA]
2.primase [synthesizes RNA primer]
4.ss binding proteins [stabilizes the strand]
2.ELONGATION OF DNA
Requires RNA primer, DNA template , DNAP
and deoxyribonucleotides [dATP,dGTP ,dCTP,
DNA polymerase catalyze the stepwise addition of
deoxyribonucleotides to 31 end of template strand
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
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.
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.
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
A small portion of 31 end of parent strand
is not replicated and length of
Telomeres play a crucial role in eukaryotic
Telomeres contain the repeat sequence of
They prevent the shorting of chromosome
with each cell division by an enzyme
Telomerase enzyme synthesizes and
maintains the telomeric DNA.
Telomerase adds repeats to 31end 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
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
E.g. actinomycin, mitomycin
2.Inhibitors of deoxy-ribonucleotides;
E.g. Anti-folates [ inhibits
3.Inhibitors of replicative enzymes;
E.g. norflox [inhibit DNA
Replication in Eukaryotic cells
More complex than prokaryotic replication
Semicoservative ,occurs bidirectional from many oigins forming multiple
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
α Nucleus Replication of nuclear DNA
Gap filling & synthesis of lagging
β Nucleus Proof reading & Repair of nuclear
Replication of mitochondrial DNA
δ Nucleus Replication of nuclear DNA
Leading strand synthesis
ε 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
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
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 )
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