Dna Replication

DNA Replication
DNA Replication
 Copying of DNA.
 Starts with one double-stranded DNA
molecule producing two identical copies
 it is the basis for
• Transfer of genetic information from
parents to the progeny
• Cell division
General Features of Replication
1. Semi-Conservative
2. Starts at Origin
3. Bidirectional
4. Semi-Discontinuous
Proposed Models of DNA Replication
• Semiconservative mode: The dsDNA

contains one parental and one daughter
strand following replication
• Conservative mode: Both parental strands
stay together after DNA replication
Semiconservative
replication:
Parental strand
remains intact
DNA Replication
Semiconservative:
During a round of replication,
each of the two strands of
DNA is used as a template for
synthesis of a new,
complementary strand.
Structure of DNA
• Eukaryotes: each chromosome contains a

long linear molecule of dsDNA
 (circular DNA in Mitochondria)
Prokaryotes: single, double

stranded, supercoiled, circular
chromosome
Steps involved in DNA replication
1. Identification of the origins of replication &
separation of 2 complementary strands
2. Formation of the replication fork
3. Initiation of DNA synthesis and
4. Elongation
Origin of Replication
• DNA replication begins at a single, unique
nucleotide sequence, the origin of replication
• include a short sequence composed almost
exclusively of AT base pairs
• Binding of DnaA protein causing local
denaturation
Separation of the two complementary
DNA strands
• binding of DnaA protein at oriC leading to
local denaturation and unwinding of AT rich
region
• dsDNA separated (melted), as polymerase use
only ssDNA as a template.
Formation of the replication fork
• unwinding and separation of two strands form,

Replication fork.
• it moves along the DNA molecule as synthesis
occurs.
• Replication of double-stranded
DNA is bidirectional.
Proteins required for DNA strand separation
1. DnaA protein
2. Single-stranded DNA-binding (SSB)

proteins
3. DNA helicases
DnaA protein
binds to specific nucleotide sequences at the

origin of replication (rich in AT base pairs). This
ATP-requiring process causes the double-stranded
DNA to melt forming localized regions of single-
stranded DNA.
Single-stranded DNA-binding (SSB) proteins
 Bind only to single-stranded DNA
• keep the two strands of DNA separate, i.e.

provide the single-stranded template
required by polymerases,
• Protection from nucleases.
DNA helicases
• bind to single-stranded DNA near the
replication fork, and then move ahead,
forcing the strands separate
• Helicases require energy provided by ATP
Effect: unwinding the double helix.

Ahead of the replication fork the DNA becomes
supercoiled.
The supercoiling
ahead of the fork
needs to be
relieved or tension
would build up and
block fork
progression.
Problem of supercoiling
• separation of two strands of the double helix:
positive supercoils in the region of DNA ahead of
the replication fork
• interfere with further unwinding of the double
helix.
• DNA topoisomerases solve the problem
DNA topoisomerases
 Type I DNA topoisomerases:
 Type II DNA topoisomerases (DNA Gyrase)
• have both nuclease (strand-cutting) and ligase
(resealing) activities.
Anticancer agents, such as Etoposide target

human topoisomerase II
DNA replication
• DNA polymerase cannot initiate synthesis on a
totally single-stranded template. Rather, they
require an RNA primer
• 3'-end of the RNA strand serves as the first
acceptor of a nucleotide by action of DNA
polymerase
RNA polymerase (primase)
• synthesizes the short stretches of RNA
(approximately 10 nucleotides long) that are
complementary to the DNA template (the U in
RNA pairs with A in DNA).
• The building blocks for this process are 5'-
ribonucleoside triphosphates (ATP, UTP, CTP,
and GTP).
Direction of DNA replication
• The DNA polymerasesIII (read parental strand

in 3’ to 5’ direction) synthesize the new DNA
strands in the 5’ to 3’ (antiparallel)
• The 2 newly synthesized stretches grow in
opposite direction
• accomplished by a slightly different
mechanism on each strand
Replication fork
β2 subunit forms a ring & allows the polymerase enzyme
to move without falling off the DNA substrate
Leading strand
• The strand that is being copied in the
direction of the advancing replication fork
• synthesized almost continuously
Lagging strand
• The strand that is being copied in the direction
away from the replication fork
• discontinuous synthesis, with small fragments
of DNA being copied near the replication fork.
These short stretches of discontinuous DNA,
termed Okazaki fragments
The looping of the template
for the lagging strand enables
DNA polymerase III to
synthesize both daughter
strands.
The leading strand is shown in
red, the lagging strand in blue,
and the RNA primers in green
Lagging strand
• Replication on lagging strand is more complex.
• Accomplished by looping of the template for the
lagging strand and Synthesized in fragments
• so that 5’ to 3’ polymerization leads to overall
growth in 3’ to 5’ direction.
• Addition of 1000 of nucleotides in a lagging
strand at a loop
• Again new loop, new stretches of RNA and finally
other okazaki fragments will be formed
Chain elongation
• DNA polymerases elongate a new DNA strand
by adding deoxyribonucleotides, one at a time,
to the 3'-end of the growing chain
• 3’-OH group of the RNA primer act as the
acceptor of the first deoxyribonucleotide
• DNA polymerase III is a highly "processive
enzyme”
Proof reading of newly synthesized DNA
• Nucleotide sequence of DNA replicated with
no errors
• Misreading of template sequence: mutation
• To ensure replication fidelity, DNA
polymerase III also has proofreading (3’ to
5’ exonuclease) activity
• eg. If the template base is C……
Excision of RNA primers and their
replacement by DNA
• DNA Polymerase I (5’→3’ exonuclease
activity) removes RNA primer hydrolytically
• Replaces it with deoxyribonucleotides
synthesizing DNA in 5’→3’direction &
• also proofreads the new chain using
5’→3’exonucleases activity.
DNA ligase
• Finally, the stretches (5'-phosphate group
and the 3'-hydroxyl group) linked by DNA

Ligases,
• Require ATP
Representation of the enzymatic events at a
replication fork
Eukaryotic DNA replication
 resembles that of prokaryotic DNA
replication
Some differences:
• Multiple origin of replication in Eukaryotes
• RNA primers removed by RNaseH
• Five classes of Eukaryotic DNA polymerases
A comparison of prokaryotic and
eukaryotic DNA polymerases
E coli Mammalian Function
I α Gap filling and synthesis of lagging
strand
II ε DNA proofreading and repair
β DNA repair
γ Mitochondrial DNA synthesis
III δ Processive, leading strand
synthesis
Proteins involved in replication
Protein Functions
Helicases Processive unwinding of DNA
SSB Prevent premature reannealing of dsDNA

Topoisomerases Relieve strain that results
from helicase-induced unwinding
DNA primase Initiates synthesis of RNA primers
DNA polymerases Deoxynucleotide polymerization
DNA ligase Seals the single strand nick between
the nascent chain and Okazaki fragments
on lagging strand
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DNA Replication

• Semiconservative mode: The dsDNA

• Eukaryotes: each chromosome contains a

Prokaryotes: single, double

2. Formation of the replication fork

3. Initiation of DNA synthesis and

• unwinding and separation of two strands form,

2. Single-stranded DNA-binding (SSB)

binds to specific nucleotide sequences at the

 Bind only to single-stranded DNA

• keep the two strands of DNA separate, i.e.

Effect: unwinding the double helix.

Anticancer agents, such as Etoposide target

• The DNA polymerasesIII (read parental strand

and the 3'-hydroxyl group) linked by DNA

SSB Prevent premature reannealing of dsDNA

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