DNA Replication

DNA REPLICATION
Schematic Overview
DNA Replication: a synopsis
 Occurs in the S phase of the cell cycle
 Replication is semiconservative, with each DNA strand
serving as template for synthesis of the complementary
strand
 DNA synthesis occurs in replicons consisting of an origin
of replication and two diverging replication forks
(bidirectional)
 Each replication fork contains a complex of enzymes,
including DNA polymerase
 Other enzymes include primase, helicase, topoisomerase
DNA replication is semiconservative
Fork movement
Replication is
semiconservative, with
each DNA strand
serving as template for
synthesis of the
complementary strand
Replication
fork
This is true for all
eukaryotes, prokaryotes,
viruses and bacteriophage
DNA Replication Hypothesis
Meselson-Stahl Experiment
The Meselson-Stahl Experiment
DNA Synthesis
Replication fork DNA chain growth
growth 5’ 3’
3’ 5’
DNA chain growth

Leading and Lagging Strands
 Limitation is imposed by synthesis of
DNA in a 5’ to 3’ direction only
 The two DNA strands are used differently
at replication fork
 leading strand is used for continuous DNA
synthesis
 lagging strand is used in discontinuous
synthesis
• forms Okazaki fragments leading
• fragments joined by DNA ligase
lagging
Lagging strand synthesis
Must supply a primer (i.e.
3’-OH) to start DNA
synthesis
This is the function of
primase which makes RNA
primers
Why RNA?
Must ‘seal’ the DNA

fragments made on the
lagging strand template
This is the function of
DNA ligase
Polymerization of DNA
 DNA polymerization requires the following:
 DNA polymerase
 dATP, dGTP, dCTP, dTTP (collectively dNTPs)
 single-stranded DNA template
 primer with free 3’ hydroxyl to accept incoming
nucleotide
 Accessory enzymes/proteins
 single-stranded DNA binding protein
 primosome containing RNA primase
 helicase to unwind DNA
 gyrase to relax supercoils formed by unwinding
DNA REPLICATION
COMPONENTS :
1. Template DNA (cetakan)
2. Origin of replication (ori)
3. Proteins :
a. DnaA, DnaB, DnaC: recognise ori and separate the strand.
b. Rep protein, SSB (single-stranded binding) protein
DnaB and Rep: Helicase
4. Nucleotides: dATP, dTTP, dGTP, dCTP
5. Enzymes:
a. Gyrase: uncoil the DNA.
b. Helicase: unwind the double helix.
c. DNA-directed RNA polymerase: synthesis of RNA primers.
d. Primase: synthesis of RNA primers.
e. DNA-directed DNA polym erase III: the actual replicating
enzyme.
f. DNA-directed DNA polymerase I:
• removes RNA primers and replace with DNA.
• proof-reading enzyme.
g. DNA ligase: joins the Okazaki fragments.
! ORI :
E. coli (and other prokaryores) has a single ori (245 bp).
Eukaryot: multiple ori.
! Helicase :
" consists of DnaB and Rep protein.
" along with SSB, separate DNA strand (breaking H bond).
• D NA- di re cte d R NA p ol a n d P rim as e :
! synthesise primer (RNA) of 2 - 10 nucleotides.
! attach to template strand for leading strand synthesis.
! primase alone attach to template for lagging strand synthesis.
• D NA- di re cte d DNA p ol III :

! the actual replicating enzyme.
! holoenzyme consists of at least seven proteins.
• D NA- di re cte d DNA p ol I :

! Kornberg enzyme.
! essential for synthesis and repair.
! 3 activities :
" DNA polymerisation.
" Exonuclease activity:
# Depolymerisation (5’ $ 3’)
# Depolymerisation (3’ $ 5’): proof-reading activity
" RNA primer removal.
• S y nt h e sis o f l ag gi n g s tr a n d :
! prokaryotes : 1000 ~ 2000 nucleotides
! eukaryotes : 100 ~ 200 nucleotides
• D NA li ga se : joins DNA molecule.
• F u n cti ons o f ba cte ri a l r e pl i co n :

% initiating a replication cycle
% controlling the frequency of initiation
% segregating replicating chromosome
• Ori in e uk ar yo te s : functions only f or replication not for

segregation.
BASI C M ECHANI SM OF DNA REP LI CATION
• R e p l ic at i on r a te : 1500 n u cl e oti d e s/sec i n E. col i
• R e p l ic at i on I n iti a ti on :
! ori is A-T rich sequence.
! ori will be functional only if it contains all s equences
specifying its authentic ori nature.
! Steps :
" DnaA binds at oriC # local melting of strand ( ~ 40 bp),
requires ATP
" Binding of DnaB and DnaC
" Helicase, SSB and gyrase begin to uncoil and un wind
DNA strands
" Attachment of RNA pol and primase to te mplate for
leading strand, and primase alone to template for lagging
strand
" Formation of replication fork
" Synthesis of leading and lagging strands
! One initiated, replication in prokaryot and eukaryot

proceeds bidirectionally from a replication bubble.
• L e a d i n g s tra nd sy nt h e sis :
! direction of synthesis (5’ $ 3’ ) coincides with direction of
replication.
! only one primer RNA is required.
! synthesis occurs continuously.
• L a ggi n g s tra nd sy nt h e sis :

! direction of synthesis is in opposite direction of replication.
! many primers are required.
! synthesis occurs discontinuously creating Okazaki
fragments (1000 ~ 2000 nucleotides).
! Th et a (") r e pl i ca ti on :
# as replication forks move bidirectionally, the circular
chromosome will resemble the theta (") structure.
# when the two replication forks merge, the double-stranded
DNA strands will then be separated by DNA gyrase.
! Te rmi n at i on of r e pl i ca ti on i n E. col i :
# termination regions : terD, terA (for replication fork 1) and
terC, terB (for replication fork 2).
# termination regions reside within ~ 100 kb on either side of
the fork meeting point.
# termination regions function as replication fork trap in case
one of the two forks moves more rapid than the other. The
more rapid fork will be t rapped at the ter region to wait for
the arrival of the slow fork.
# ter sequence : ~ 23 bp.
# termination of replication requires tus protein which
recognise ter consensus sequence and prevents replication
fork from proceeding.
REPLI CATION OF EUKARYOTIC CHROM OSOM E
• Eukaryotic chromosome contains many replicons.

• Multiple ori, spaced about 20 kbp apart.
• The decision to replicate DNA is made in the G1 phase of the
cell cycle, but the replication itself occurs at S phase.
• At least 5 types of DNA polymerase.
• Slower (6 - 8 hours) than prokaryotic DNA replication (~ 40
minutes).
• Bidirectional.
• Replication of the end of chromosome (or linear replicon):
$ primed by a protein covalently linked to 5’ end
Replication Fork Formation and Movement
Replication Machine
Role of primase? Role of helicase?

Role of topoisomerase? Role of single-stranded DNA binding proteins?
Replication Fork Formation and Movement
Direction of replication
Origins of DNA Replication
 DNA replication begins from specific nucleotide

sequences called origins of replication
 recognized by origin recognition proteins that open
the helix and recruit the replication machinery
 DNA synthesis proceeds in both directions
outward from the origin
 replicated double helices being produced ultimately
join each other
 when complete, there are two identical daughter
molecules
Bidirectional DNA Replication
DNA synthesis occurs in replicons consisting of an origin
of replication and two diverging replication forks
(bidirectional)
Fork movement Fork movement

The structure of replication
terminator in E. coli
Replication of Circular Chromosome
• Replication begins from a

single origin of replication
• Replication is bidirectional
–intermediate is called a
theta structure
Theta replication
Rolling Circle Replication
Mode of replication used by
some plasmids and some phage
One strand remains circular and
acts as template for continuous
synthesis
Displaces the 5’-end of the strand
which acts as template for
discontinuous synthesis
Synthesis often continues beyond
unit length to form a multimer of
head-to-tail copies of the genome
Cut and joined to form new
circular molecules
Rolling-circle replication
Special cases
 Some circular DNA molecules (e.g., plasmids)
replicate by rolling circle model
 continuous replication from circular template
 discontinuous replication (Okazaki) from displaced
single strand
 Telomeres
 specialized ends of linear eukaryotic chromosomes
 replicated by telomerase
• carries small template RNA
• a type of reverse transcriptase
 without telomerase, lagging strands continuously
shorten with removal of primer, eventually deleting
essential genes
Replication of ΦX 174
TMV Replication (RNA replication)
Replication of Retrovirus
Telomere replication in Tetrahymena

DNA Replication

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DNA REPLICATION

DNA chain growth

Must ‘seal’ the DNA

1. Template DNA (cetakan)

2. Origin of replication (ori)

b. Rep protein, SSB (single-stranded binding) protein

DnaB and Rep: Helicase

4. Nucleotides: dATP, dTTP, dGTP, dCTP

• D NA- di re cte d DNA p ol III :

• D NA- di re cte d DNA p ol I :

• D NA li ga se : joins DNA molecule.

• F u n cti ons o f ba cte ri a l r e pl i co n :

• Ori in e uk ar yo te s : functions only f or replication not for

• R e p l ic at i on r a te : 1500 n u cl e oti d e s/sec i n E. col i

! One initiated, replication in prokaryot and eukaryot

• L a ggi n g s tra nd sy nt h e sis :

REPLI CATION OF EUKARYOTIC CHROM OSOM E

• Eukaryotic chromosome contains many replicons.

Role of primase? Role of helicase?

 DNA replication begins from specific nucleotide

Fork movement Fork movement

• Replication begins from a

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