DNA

Replication
DNA – DOUBLE
HELICALSTRUCTURE
WATSON and CRICK-
Model
[Link]
DNA – DOUBLE
HELICALSTRUCTURE
DNA REPLICATION :
Salient Characteristics
1) Semiconservative
2) Bidirectional and semi discontinuous
3) 5’ 3’ direction
4) Proof reading
5) DNA polymerase
6) Enzyme and protein involved in DNA replication
Directionality of DNA
Nucleotides in DNA
backbone are bonded
together by phosphodiester
linkage between 3′&
5′carbons.

DNA molecule has “direction.“

Complementary strands run

in opposite directions.
DNA Replication-
Introduction
Basis for inheritance

Fundamental process occurring in

all cells for copying DNA to transfer
the genetic information to daughter
cells
Each cell must replicate its DNA
before division.
DNA
Replication
Semi conservative

Parental strands are not

degraded
Base pairing allows each
strand to serve as a template
for a new strand
New duplex is 1/2 parent
template & 1/2 new DNA
 Okazaki

DNA
Replication
Semi discontinuous
Leading & Lagging
strands
Leading strand
continuous synthesis
Lagging strand
Okazaki fragments
joined by ligases
DNA
Replication
Energy of Replication
The nucleotides arrive as
nucleoside triphosphates
DNA base, sugar with PPP
○ P-P-P = energy for bonding

DNA bases arrive with their own energy source

for bonding
bonded by enzyme: DNA polymerase III
DNA
Replication
Primer is needed
DNA polymerase can only add nucleotides
to 3′end of a growing DNA strand
○ need a “starter” nucleotide to make a
bond
strand only grows 5′→3′.
Template is read in the 3′-5′direction while
polymerization takes place in the 5′→3′
direction
Primer
RNA primer
Synthesized by Primase
serves as a starter sequence for DNA
polymerase III
Only one RNA Primer-required for the
leading strand
RNA Primers for the lagging strand depend on
the number of “OKAZAKI FRAGMENTS”
RNA Primer has a free 3’OH group to
which the first Nucleotide is bound.
Origin of
replication

Local unwinding
DNA

Formation of
Replication Bubble
 Replication fork

Leading Lagging
strand strands

Continuous manner Discontinuous manner

5’ 3’towards fork 5’3’away from fork
Single RNA primer Multiple primers required
Multiple polymerase required
DNA
Replication-Steps
Identification of the origins of replication
Unwinding (denaturation) of dsDNA to provide
an ssDNA template
Formation of the replication fork
Initiation of DNA synthesis and elongation
Primer removal and ligation of the newly
synthesized DNA segments
Reconstitution of chromatin structure
Components of Replication
DNA polymerases- Deoxynucleotide
polymerization
Helicase -Processive unwinding of DNA
Topoisomerases Relieve torsional strain
that results from helicase-induced
unwinding
RNA primase Initiates synthesis of RNA
primers
Single-strand binding proteins Prevent
premature reannealing of dsDNA
DNA ligase Seals the single strand nick
between the nascent chain and Okazaki
fragments on lagging strand
Originof Replication-
Prokaryotes
At the origin of replication (ori), there is an
association of sequence-specific dsDNA-
binding proteins with a series of direct
repeat DNA sequences.
In E coli, the oriC is bound by the protein
dnaA.
a complex is formed consisting of 150–250
bp of DNA and multimers of the DNA-
binding protein. This leads to the local
denaturation and unwinding of an adjacent
A+T-rich region of DNA.
Origin of Replication -Eukaryotes
Functionally similar autonomously replicating
sequences (ARS) or replicators have been identified
in yeast cells.

The ARS contains a somewhat degenerate 11-bp

sequence called the origin replication element (ORE).

The ORE binds a set of proteins, analogous to the

dnaA protein of E coli, which is collectively called the
origin recognition complex (ORC).

The ORE is located adjacent to an approximately 80-bp

A+T-rich sequence that is easy to unwind. This is
called the DNA unwinding element (DUE).
Unwinding of
DNA
The interaction of proteins with ori
defines the start site of replication and
provides a short region of ssDNA
essential for initiation of synthesis of
the nascent DNA strand.
DNA Helicase allows for
processive unwinding of DNA.
Single-stranded DNA-binding
proteins (SSBs) stabilize this
complex.
In cooperation with SSB, this leads
to DNA unwinding and active
Unwinding of
DNA
Formation of the
Replication Fork
The polymerase III holoenzyme binds to
template DNA as part of a multiprotein
complex
DNA polymerases only synthesize DNA in
the 5' to 3' direction,
Because the DNA strands are antiparallel ,
the polymerase functions asymmetrically.
On the leading (forward) strand, the DNA
is synthesized continuously.
On the lagging (retrograde) strand, the DNA
is synthesized in short (1–5 kb)fragments,
the so-called Okazaki fragments.
Replication
Fork
Formation of Replication
Bubbles
Replication occurs in both directions
along the length of DNA and both
strands are replicated
simultaneously.
This replication process generates
"replication bubbles"
Replication
Bubbles
The DNA Polymerase
Complex
A number of different DNA polymerase
molecules engage in DNA replication.
These share three important properties: (1)
chain elongation, (2) Processivity, and
(3) proofreading.
Chain elongation accounts for the rate (in
nucleotides per second) at which
polymerization occurs.
Processivity is an expression of the number of
nucleotides added to the nascent chain before
the polymerase disengages from the template.
The proofreading function identifies copying
errors and corrects them
DNA Polymerase
Complex
In E coli, polymerase III (pol III)
functions at the replication fork. Of all
polymerases, it catalyzes the highest
rate of chain elongation and is the most
processive.
Polymerase II (pol II) is mostly involved
in proofreading and DNA repair.
Polymerase I (pol I) completes chain
synthesis between Okazaki
fragments on the lagging strand.
Differences between DNA
Polymerase I, II and III
Eukaryotic DNA
polymerases
Eukaryotic cells have counterparts for
each of these enzymes plus some
additional ones. A comparison is
shown in Table-
Initiation & Elongation of DNA
Synthesis
Primer-The priming process involves the
nucleophilic attack by the 3'-hydroxyl
group of the RNA primer on the
phosphate of the first entering
deoxynucleoside triphosphate with the
splitting off of pyrophosphate.
Mammalian DNA polymerase Alpha is
mainly responsible for the synthesis
of primer.
Initiation & Elongation of DNA
Synthesis
Selection of the proper
deoxyribonucleotide whose terminal 3'-
hydroxyl group is to be attacked is
dependent upon proper base pairing
with the other strand of the DNA
molecule according to the rules
proposed originally by Watson and
Crick
Initiation & Elongation of DNA
Synthesis
When an adenine deoxyribonucleoside
monophosphoryl moiety is in the template
position, a thymidine triphosphate will enter
and its phosphate will be attacked by the 3'-
hydroxyl group of the deoxyribonucleoside
monophosphoryl most recently added to the
polymer.
By this stepwise process, the template dictates
which deoxyribonucleoside triphosphate is
complementary and by hydrogen bonding
holds it in place while the 3'-hydroxyl group of
the growing strand attacks and incorporates
the new nucleotide into the polymer.
Base pairing in DNA
Replication
DNA Topo
isomerases
Relief of super coils is dome by
Topo isomerases
Two types:
Topoisomerases I : acts by making a transient
single cut in the backbone of the DNA, enabling
the strands to swivel around each other to
remove the build-up of twists
Topoisomerase II (DNA Gyrase) acts by
introducing double standed breaks enabling one
double-stranded DNA to pass through another,
thereby removing knots and entanglements that
can form within and between DNA molecules.
Formation of super coils
Mechanism of action of Topo
isomerases(Type-I and
Type-II)
Primer removal and Nick
sealing
Primers are removed by DNA
polymerase I by replacing
ribonucleotides with deoxy
Ribonucleotides
Nicks are sealed by DNA ligase
Multiple primers on the Lagging
strand while single primer on the
leading strand.
Proof reading and Editing
1000 bases/second =
lots of typos!
DNA polymerase I
proofreads & corrects typos
repairs mismatched bases
removes abnormal bases
○ repairs damage
throughout life

reduces error rate from

1 in 10,000 to
1 in 100 million bases
Terminationof replication

In prokaryotes:
DNA replication terminates
when replication forks reach
specific “termination sites”.
the two replication forks meet each
other on the opposite end of the
parental circular DNA .
Terminationof replication
This process is completed in about 30
minutes, a replication rate of 3 x 105
bp/min in prokaryotes
The entire mammalian genome
replicates in approximately 9 hours, the
average period required for formation
of a tetraploid genome from a diploid
genome in a replicating cell.
Summary of
Replication
Unwinding- forms replication fork
Primase- Synthesizes RNA primer
Continuous synthesis -Leading strand
Discontinuous synthesis – Lagging
strand (Okazaki fragments)
Synthesis 5’-3’ direction
Primers removed, nick sealed
Proof reading by DNA
polymerases Organized in to
chromatin structure