DNA

Replicatio
n Vipin Shankar
In the early 1930s, biologists began
speculating as to what sort of
molecule could have the
kind of stability that the gene
demanded,
yet be capable of
permanent, sudden change to the
mutant forms
that must provide
What is the genetic material?

• Is it
– The proteins, that make up the
enzymes?
– The complex proteins of the
chromosomes?
– The amino acids that make up the
proteins?
– Or, the seemingly simple nucleic acids
that make up the chromosomes?
Avery’s Bombshell

• Oswald T Avery, Colin M MacLeod and

Maclyn McCarty (Rockefeller Institute,
New York), based on original
observations by Griffith.
• DNA can carry genetic specificity.
The Double Helix
The Cell Cycle
Replication

• A template directed nucleic acid

synthesis reaction.
• Replication leads to doubling of the
DNA, preserving the genetic
information, for transmission to the
next generation.
• Occurs in the S phase of the Cell
Cycle.
• Replication requires a template to
provide sequence information.
DNA Replication – The possible
mechanisms.

“… It has not escaped our notice that

the specific pairing we have
postulated immediately suggests a
possible copying mechanism for the
genetic material….”

- Watson and Crick

(in the paper describing DNA structure)
The possible mechanisms…

• The Conservative model

– Both parental strands remain together
and the two new strands of DNA would
form an entirely new DNA molecule.

• The Dispersive model

– The strands get broken as frequently as
ten base pairs and are used to prime
the synthesis of similarly short regions
of DNA, which get subsequently joined
to form the complete DNA strand.
The possible mechanisms…

• The Semi-conservative model

– The two strands separate during
replication and each strand act as the
template for a new strand.
– Thus the new DNA molecule is made up
of a newly synthesized strand and a
strand from the original molecule.
Experimental evidence for strand
separation during replication
• Mathew Meselson and Frank Sthal
(1958), at the California Institute of
Technology.
The Meselson - Sthal experiment
• They grew E. coli in a medium
containing 15NH4Cl as the only source
of nitrogen.
• After growing for several generations,
on the 15N-media, the DNA was found
to be denser.
• The density of the strands were
determined by CsCl-density gradient
centrifugation.
• Meselson and Sthal, transferred the E.
coli, with the heavy (15N) DNA, to a
media containing 14NH4Cl as the only
More proof for semi-conservative
replication
• Taylor et al, labeled Vicia fava
(broad bean) root tip cells with
3
H-thymidine and allowed
them to grow in unlabelled Dr. J Herbert
medium. Taylor

• The metaphase chromosomes

were analyzed by
autoradiography.
• Observations:
– Both chromatids were labeled
after one generation.
• Chromosome from parent cell labeled with 3H-
thymidine.
• Cells grown in medium without 3H-thymidine.
• Chromatids separate during cell cycle. And each
chromatid produces its sister chromatid
• The newly produced chromatids are not labeled.
More proof…

• Based on the use of 5-

bromodeoxyuridine (BrdU), an
analogue of thymidine.
• DNA with BrdU in place of thymidine,
does not stain with fluorescent dye
(33258 Hoechst).
• When cells labeled with BrdU are
subsequently grown in a medium
without the analogue
– Only one chromatid takes up the stain
while its sister does not.
Dr. Cairns’ Experiment

• Dr. J Cairns (1963) used

autoradiography to demonstrate
semi-conservative model of
replication.
• He grew E. coli on a medium
containing 3H-thymine.
• The DNA was then extracted and
carefully subjected to
autoradiography.
Dr. Cairns experiment: inferences

• The E. coli DNA is a circle.

• The DNA is replicated while
maintaining the integrity of the circle.
• An intermediate theta structure forms
(topologically similar in shape to the
Greek letter ‘θ’.)
• Replication of the DNA seems to be
occurring at one or two moving Y-
junctions in the circle.
• The DNA is unwound at a given point,
and replication proceeds at a Y-
junction, in a semi-conservative
The rolling circle replication

• This form of replication is initiated by a

break in one of the nucleotide strands that
creates a 3’-OH group and a 5’-phosphate
group.
• New nucleotides are added to the 3’end of
the broken strand, with the inner
(unbroken) strand used as a template.
• As new nucleotides are added to the 3’end,
the 5’end of the broken strand is displaced
from the template, rolling out like thread
being pulled off a spool.
• The 3’end grows around the circle, giving
rise to the name rolling-circle model.
The rolling circle mode…

• The replication fork may continue around

the circle a number of times, producing
several linked copies of the same sequence.
• With each revolution around the circle, the
growing 3’ end displaces the nucleotide
strand synthesized in the preceding
revolution.
• Eventually, the linear DNA molecule is
cleaved from the circle, resulting in a
double stranded circular DNA molecule and
a single-stranded linear DNA molecule.
• The linear molecule circularizes either
before or after serving as a template for the
The replicon

• Stretches of DNA with a single origin

of replication (Francois Jacob, Sydney
Brenner & Jacques Cuzin (1963)).
• The entire DNA replicated from a
single origin.
• 2 components:
– The initiator
– The replicator
The replicon…

• The initiator
– A protein that specifically recognize the
replicator.
– Recruits the replication machinery to
the origin of replication.
• The replicator
– The entire set of DNA sequences
sufficient to direct the initiation of
replication.
– Composed of two parts
• A recognition site for the initiator
The origin of replication

• A stretch of AT rich DNA, that unwinds

readily but not spontaneously.
• Called oriC in E. coli.
– Contains 2 repeated motifs.
– A 9-mer motif repeated 5 times &
– A 13-mer motif repeated 3 times.
The mechanism of replication
• Tightly controlled process,
– occurs at specific times during the cell
cycle.
• Requires:
– A set of proteins and enzymes,
– Energy.
• Two basic steps:
– Initiation
– Elongation.
• Two basic components:
– Template
– Primer.
Process of semi-conservative
replication
• Identification of the origins of
replication.
• Unwinding (denaturation) of ds-DNA
to provide an ss-DNA template.
• Formation of the replication fork.
• Initiation of DNA synthesis and
elongation.
• Formation of replication bubbles with
ligation of the newly synthesized DNA
segments.
Enzymology of DNA replication

• DNA Helicase: Unwinds DNA ahead of

replication fork
• DNA Polymerase: DNA synthesis and
repair of gaps
• DNA Ligase: Joins fragments of DNA
• DNA Primase: Syntheses primers
• DNA Topoisomerases: Releases
torsional strain caused by helicase
activity
• SSBs: Stabilizes single stranded
DNA Polymerases

• First identified in lysates of E. coli by

Arthur Kornberg (1963).
• The first polymerases to be isolated
was named DNA Pol – I
• Isolation of polymerases represent a
landmark discovery in molecular
biology, since the ability of these
enzymes to accurately copy a DNA
template provided biochemical basis
of the mode of replication provided by
Watson & Crick.
DNA Polymerases: more studies

• Cultures of E. coli were treated with

chemical mutagens.
• Mutants deficit in Pol-I activity were
isolated and sub-cultured.
• These strains grew normally in normal
media.
• But, these strains were extremely
sensitive to agents that cause DNA
damage.
DNA Polymerases: more studies…

• Inferences:
– Pol-I may not be required for DNA
replication.
– Pol-I may be involved in the repair of
DNA damage, rather than in DNA
replication.
DNA Polymerases: further studies

• DNA polymerases II & III were isolated

from E. coli.
• The potential role of these enzymes
were studied by the isolation of
appropriate mutants.
• Its been confirmed that
– Pol-III & Pol-I are the major enzymes
responsible for replication.
– Pol-II is responsible for ‘error-prone’
DNA repair.
• Eukaryotic cells contain 5 different
DNA Polymerases: Types
DNA Polymerases: Functions
DNA Polymerase: Mechanism

• Requirements:
– All 4 dNTPs; viz, dATP, dCTP, dGTP &
dTTP.
– A primer – template junction.
• The new chain is synthesized by
adding appropriate dNTPs at the 3’
end of the primer at the primer –
template junction.
• A phosphodiester bond is formed
between the 3’OH of the primer and
the α-phosphate group of the
DNA Polymerases: Mechanism…
• Hydrolysis of the pyrophosphate
drives the reaction.

XTP + (XMP)n → (XMP)n+1 + P ~ P

P ~ P → 2Pi

XTP + (XMP)n → (XMP)n+1 + 2Pi

DNA Polymerases: Mechanism…

• Have a single active site to catalyze

the addition of all four dNTPS.
• This is done by exploring the nearly
identical geometry of the A:T & G:C
base pairs.
• The DNA polymerase monitors the
ability of the incoming nucleotide to
form a correct base pair.
• Only when a correct base pair is
formed a phosphodiester bond is
formed between the 3’OH of the
DNA Polymerase: Mechanism…

• Incorrect base-pairing leads to lower

rates of nucleotide addition, due to
catalytically unfavorable alignment of
the substrates : Kinetic selection.
• The rate of incorporation of an
incorrect nucleotide is 10,000 fold
slower.
• DNA polymerase can distinguish
between rNTPs and dNTPs, by steric
exclusion.
DNA Polymerase: Mechanism…

• DNA Polymerase resembles a Hand

that Grips the Primer : Template
junction
• 3 domains –
– The palm:
• contains the primary elements of the
catalytic site.
• Has 2 divalent metal ions – Mg2+ or
Zn2+.
• Catalysis of the phosphodiester bond.
• Monitors the accuracy of base
DNA Polymerase: Mechanism…

• Domains
– The Fingers:
• Bind to the incoming nucleotide.
• Once the correct base pair is formed,
the finger domain moves and enclose
the dNTP, thus enhancing catalysis.
– The Thumb:
• Not intimately involved in catalysis.
• Maintains the correct position of the
primer in the active site.
• Maintains a strong association
between the polymerase and the
Processivity of DNA Polymerase

• Processivity is a characteristic of
enzymes that operate on polymeric
substrates.
• DNA polymerases are capable of
adding as many as 1,000 nts per
second.
• The degree of processivity is defined
as the average number of nucleotides
added each time the enzyme binds a
primer : template junction.
• Each DNA polymerase has a
Processivity….

• The initial binding of the polymerase

to the primer : template junction is
the rate limiting step.
• The processivity of DNA Pol is
increased by a Sliding Clamp.
• The Sliding clamp is an association of
proteins that assemble in the shape
of a doughnut.
• The clamp encircles the newly
synthesized ds DNA and keeps DNA
Pol associated with the primer :
Proof Reading by Polymerase

• Mediated by nuclease activity that

remove the incorrectly base paired
nucleotides.
• The 3’ to 5’ exonuclease activity of
the palm domain, checks for the
incorrect base pairing.
• The proof reading gives a second
chance to correct mistakes and to
add the correct nucleotide.
DNA Replication: The process

• The initiator protein recognizes and

binds to the replicator sequence.
• The initiator protein recruits DNA
Helicase to the Origin of replication.
• DNA Helicase unwinds the ds DNA at
the origin of replication – formation of
the Replication Fork.
• Other proteins of replication
machinery assemble at the
replication fork.
DNA Replication: The process…

• Single Strand Binding Proteins (SSBs)

stabilize the replication fork by
binding to the newly opened ss DNA,
preventing the recoil.
• Topoisomerases prevent the super-
coiling formed during helicase
activity.
– 2 classes of topoisomerases are
present.
– Class II topoisomerases also called
Gyrases are the important ones in DNA
The need for
topoisomerases
DNA Replication: The process…

• Primase synthesizes RNA primer for

the action of DNA polymerase.
• PROBLEM.
– DNA Polymerase can synthesize DNA
only in the 5’ to 3’ direction.
– So synthesis on one strand is
continuous.
– What happens on the other strand?
• The strand on which continuous DNA
synthesis proceeds is called the
Leading Strand.
DNA Replication: The process…

• The second strand is called the

Lagging Strand.
• On the lagging strand DNA synthesis
happens in short fragments (1000 –
2000 nts in bacteria and 100 – 400 in
Ek.cells) called Okazaki Fragments.
• Each Okazaki fragment requires a
new primer.
• The RNA primers are removed by
RNAase H.
• The single stranded nicks produced
DNA Replication: Trombone Model

• At the replication fork, the leading

strand and the lagging strand are
synthesized simultaneously.
• This limits the amount of ss DNA
present in the cell during replication.
• To co-ordinate the replication of both
the strands, multiple DNA
Polymerases act together.
• A large multi-protein complex called
the DNA Pol –III holoenzyme in which
the core enzyme is associated with
Finishing replication

• Completion of replication is different in

circular and linear chromosomes.
• In a circular chromosome (like in E. coli), the
2 replication forks meet at a region called
the termination region or ter region.
• Termination utilization substance (tus
protein), forms a complex with the ter
region and stops the progress of the
replication fork.
• This results in two daughter molecules
linked to one another (Catenane).
• These are seperated by the action of class II
Replication of circular
chromosomes
Finishing Replication…

• The requirement of a RNA primer for

the synthesis of all Okazaki fragments
on the lagging strand, creates a
dilemma for the replication of the end
of the linear chromosome – the end
replication problem.
• Once RNA molecule is removed from
the last Okazaki fragment, a short
region of un-replicated DNA will
remain on the lagging strand.
• This means that each round of
replication would result in the
Telomerase

• Telomerase is a DNA polymerase that

does not require a exogenous
template.
• Telomerase is an enzyme which
includes both protein and RNA
components (ribonucleoprotein).
• The RNA component acts as a
template for adding the telomeric
sequence to the 3’ terminus at the
end of the chromosome and thus
solves the end replication problem.
The End Replication
Problem
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
nucleosomes