DNA Replication

Presented By:
K.Dharan Kumar
PharmD 3rd year
Balaji Institute
Pharmaceutical sciences
DNA REPLICATION
DNA Replication is the
biological process that occurs in
all living organisms.
It is basis for biological inheritance .
Replication is the process of synthesis of
daughter DNA from parental DNA.
Occurs in S phase of cell cycle.
Methods of DNA replication
 Conservative replication:
 The parental double helix remains intact;
 both strands of the daughter double helix are
newly synthesized.
 Semiconservative Replication
Half of the parental DNA molecule is conserved in each
new double helix,paired with a newly synthesized
complimentary strand.This is called semiconservative
Replication.
 Dispersive replication:
 At completion, both strands of both double helices
contain both original and newly synthesized material.
 Complementary base pairing produces semiconservative
replication.
 Begins with the unwinding of the double helix to expose the
bases in each strand of DNA.
 Double helix unwinds.

 Each strand acts as template.

 Complementary base pairing ensures that T signals

addition of A on new strand, and G signals addition of
C.

 Two daughter helices produced after replication.

Prokaryotic DNA replication
Basic requirements for replication
 a set of proteins and enzymes,
 and requires energy in the form of ATP.

 Basic steps:
 Initiation
 Elongation.
 Termination

 Two basic components:

 template
 primer.
Replication Enzymes & proteins
DNA Polymerase : Matches the correct nucleotides then joins /polymerizes adjacent
nucleotides to each other.
All of them possess the following biological activity
1.5'- 3' polymerase activity
2.Exonuclease activity
a.5'-3' removes primer or excise mutated segment
b.3'-5' excise mismatched nucleotides

DNA polymerase-I:Mainly responsible for proofreading and filling the

gaps, repairing DNA damage
DNA polymerase-II:Temporarily functional when DNA-Pol I and DNA-Pol III are not functional
It is capable for doing synthesis on the damaged template
Involves in DNA repair process
DNA Polymerase-III:A heterodimer enzyme consist of 10 different sub units
Enzyme responsible for elongation process.
Nucleotides are always added to the growing strand at the 3' end
 Helicase:Unwinds the DNA and melts it.
Also referred to as DnaB.It opens the double strand DNA with
consuming ATP The opening process with the assistance of DnaA and DnaC.
Primase:Provides an RNA primer to start polymerization.
It is also called DnaG
Primers are short RNA fragments of a several nucleotides long.
Primase,DnaB,DnaC and an origin form a Primosome complex at
the intiation phase.
Single strand binding Proteins:keep the DNA single stranded after it
has been melted by helicase.
SSB protein maintains the DNA template in the single strand form in
order to ✓ prevent the dsDNA formation
✓ Protect the vulnerable ssDNA from nucleases
 Gyrase:A topisomerase that reliveves torsional strain in the DNA molecule.
It cuts phosphoester bonds on both strands of dsDNA,releases the
supercoil constraint and reforms the phosphodiester bonds.
It can change dsDNA into the negative supercoil state with consumption of ATP.
Ligase:joins adjacent DNA strands together (fixes nicks).
Connect two adjacent ssDNA strands by joining the 3'-OH of one DNA strand to
the 5'-P of another DNA strand.
Sealing the nick in the process of replication,repairing,recombination and splicing.
Telomerase:Finishes of ends of DNA strands in Eukaryotes.
The eukaryotic cells use telomerase to maintain the integrity of DNA telomere.
The telomerase composed of:
Telomerase RNA
Telomerase association protein
Telomerase reverse transcriptase
It is able to synthesize DNA using RNA as
template  DNA replication process
The replication process starts from the origin and proceeds
in two opposite directions .
 Stages of DNA
replication:
Initiation:
 Occurs at the origin of
replication.
 Separates dsDNA,primer
synthesis.
 Elongation:
 Involves the addition of new
nucleotides (dNTPs) based on
complimentary of the
template stran.d
 Forms phospoester bonds,
correct the mismatch bases,
extending the DNA strands.
 Termination:
 Stops DNA replication occurs
at a specific termination site.
Initiation:
The replication starts at a particular site called Origin
of replication or Ori-site.
 Origin of replication:
 Replication proceeds in both directions
(bidirectionally) from a single origin of replication on
the prokaryotic circular chromosome.
 Replication proceeds in both directions
(bidirectionally) from hundreds or thousands
of origins of replication on each of the linear
eukaryotic chromosomes.
 Bacteria have 1 origin of replication
per one chromosome.
 They only have one chromosome = 1 origin!
 Formation of Replication fork:
 DnaA recognises Ori-C.
 DnaB(Helicase) and DnaC join the DNA-DnaA complex,open the AT rich
local region,move on the template downstream further to separate enough
space.
 DnaA is replaced gradually.
 SSB protein binds the complex to stabilize ssDNA.
Replication Fork Origin of Rep
 Primer synthesis:
 Primase joins and forms a complex called primose.
 Primase starts the synthesis of primers on the ssDNA template using NTP as
the substrates in the 5'-3' direction at the expense of ATP.
 The short RNA fragments provide free 3'-OH groups for DNA elongation.

 Elongation:
 dNTPS are continuously connected to the primer or the nascent DNA chain by
DNA-pol III.
 The core enzymes catalyse the synthesis of leading and lagging
strands Respectively.
 The nature of the chain elongation is the series formation of
the phosphodiester bonds.
 Lagging strand synthesis:
 RNA primers on OKAZAKI fragments Are digested by the enzyme
RNAase.  The gaps are filled by DNA -pol I in the 5'-3' direction.
 The nick between the 5' end of one fragment and the 3' end of the next fragment
is sealed by ligase .
 OKAZAKI fragments:
 Many DNA fragments are synthesized sequentially on the DNA template
strand having the 5'-end.These DNA fragments are called "OKAZAKI
fragments“.
 They are 1000-2000 nt long in Eukaryotes.
 The daughter strand consisting of OKAZAKI fragments is called
the lagging strand.

 Termination:
 The replication of E.coli is bidirectional from one origin and two replication
forks must meet at one point called ter at 32.
 All the primers will be removed and all the fragments will be connected by DNA-
pol 1 and ligase.
 Ter binding proteins:will recognises the termination
sequences and helps to achieve the termination process.
Eukaryotic DNA replication
Basic requirements for replication
 a set of proteins and enzymes,
 and requires energy in the form of ATP.

 Basic steps:
 Initiation
 Elongation.
 Termination

 Two basic components:

 template
 primer.
Enzymes and proteins of DNA replication
proteins Function
 Dna A protein  Recognizes ori sequences
 Dna B protein  Unwinds/opens dsDNA
(DNA Helicase)
 Dna C protein  Assists Dna B to bind at ori-site
 DNA polymerases  Synthesizes the new DNA strands
 Dna G protein  Synthesize RNA primer
(DNA primase)
 Single  Binds single-stranded DNA
strand binding proteins(SSB)  DNA  Relieves torsional strain generated
by unwinding
Gyrase
(DNA Topoisomerse)
Eukaryotic DNA replication
DNA replication closely related with
cell cycle.
Multiple origins on chromosome and
replication are activated on a
sequential order rather
than simultaneously.
DNA Polymerse of Eukaryotes

Eukaryotic Enzyme Prokakaryotic Enzyme

DNA-pol α:initiate DnaG,primase
replication and synthesize
primers. Repair
DNA-pol β:Replication with
low fidelity.
DNA-pol γ:Mitochondrial DNA
synthesis.
DNA-pol δ:elongation . DNA-pol III

DNA-pol ε:lagging strand synthesis,

proofreading and gap filling. DNA-pol I
INITIATION
The eukaryotic replication origins are shorter than that of E.coil.that ori-
sites in eukaryotes called ARS(Autonomously
Replicating Sequences)/Replicators.
Requires DNS-pol α (primase activity)and DNA-pol δ (polymerase activity and
helicase activity).
DNA –pol δ requires a protein called for it s activity
Proliferating cell Nuclear Antigen(PCNA).
Need Topoisomerase and Replication factors(RF) to assist.
 DNA origin of replication.
 Initiator proteins bind
 Recruits DNA
 helicase
 Opening of DNA strands.
 Replication Initiation:
 Primase and the RNA Primer

 Replication Elongation:
 DNA polIII
 Must have 3’ to add to

 Replication is Finished:
 DNA polI removes primer

 Fills gap using 3’ends

 DNA ligase connects

frags  Uses 5’ ends!
 Elongation:
DNA replication and nucleosome assembling
occur simultaneouslyOverall replication speed
is compatible with that of prokaryotes.

DNA pol works as a dimer Lagging strand

must
loop around to accommodate dimerization.

Origin of Rep
Termination:
 The ends of chromosomes (telomeres) cannot be replicated on the lagging strand because there is no
primer available.

 Telomerases

 enzymes that contain RNA primers which extend the ends of chromosomes (not
normally expressed in significant levels).
 Telomeres form a sort of single stranded cap around the chromosome ends to protect
them from being degraded.
 chromosome ends are progressively shortened with each round of
replication.  “old” cells with shortened telomeres undergo apoptosis -
 Protective for normal cells.
 Kill the old and possibly mutated.

 Telomerase is over expressed in cancer cells.

 Hypothesis is that cancer cells do not undergo apoptosis because their telomeres do not
shorten over time.
Replicating the Ends of Chromosomes
 telomerase adds an RNA primer complementary to
telomere sequences
 chromosomal replication proceeds by adding to the 3’ end of the primer.

 Fills the gap left behind by replication.

 Telomerase enzyme can also add DNA basepairs to the TEMPLATE

 complementary to the RNA primer basepairs.

 Using an RNA template to make DNA, telomerase functions as a

reverse transcriptase called TERT (telomerase reverse transcriptase).
 This goes against the Central Dogma….
 Evolutionarily thought to be derived from a Retrovirus.
Assembling Newly Replicated DNA into
Nucleosomes
 When eukaryotic DNA is replicated, it complexes with histones.
– This requires synthesis of histone proteins and assembly of new nucleosomes.

 Transcription of histone genes is initiated near the end of G1 phase,

and translation of histone proteins occurs throughout S phase.

 Assembly of newly replicated DNA into nucleosomes is shown in Figure 11.16.

The Assembly of Nucleosomes after
Replication