DNA Replication

DNA Replication

Each time a cell divides into two daughter cells, all the DNA moleculem be duplicated.
The process by which a DNA molecule produces its identical copies is described as
DNA replication.

It is a type of cell duplication or self reproduction of DNA, where two daughter
molecules are formed from a single DNA molecule.

Theoretically, three possible modes of DNA replication are possible. They are:

1. Dispersive replication:

The two strands of parent DNA break
randomly and produce several Pieces. These
pieces replicate and reunite to form new
daughter DNA molecules. These new DNA molecules
contain a mixture of old and new nucleotides
scattered along, the chains. The daughter molecules
can be described as hybrids. This mechanism is now

accepted nor proved experimentally.

2. Conservative replication:

After replication, one daughter DNA contains the original
two strands of the parent molecule. While the other
daughter molecule contains two newly synthesized
strands.this method is also not accepted.

3. Semi-conservative replication:

This method of DNA replication was proposed by Watson
and Crick. Because of specificity of base pairing, the
sequence of bases along one chain automatically
determines the basesequence along the other.

Half of the DNA is conserved i.e., only one strand is
synthesized and the other half of the original DNA
is retained.

The evidence for semi conservative replication of DNA molecules were provided by
Meselson and Stahl using ¹⁵N - a heavy isotope of ¹⁴N. 

J Cairns demonstrated the semi – conservative mode of replication of bacterial
chromosome using autoradiography technique.

Enzymes required for Replication:

Topoisomerase:

These enzymes can change the topological form or shape of DNA. Responsible
for initiation of the unwinding of the DNA. The tension holding the helix in coiled
and supercoiled structure can be broken by nicking a single strand of DNA.
DNA topoisomerases introduce a nick on only one of the DNA strands. This allows
the molecule to roatate around the phosphodiester bond on the opposite strand
as if it were a swivel. The topoisomerases introduce negative supercoils and relieve
strains in the double helix at either end of the bubble.

The Helicase:

Unwinds and unzips the DNA helix by breaking the hydrogen bonds between the
base pairs, thus allowing the two strands to separate.
The two strands very much want to bind together because of their hydrogen
bonding affinity for each other, so the helicase activity requires energy
(in the form of ATP) to break the strands apart.

DNA Polymerase:

Chief enzyme of DNA replication. Discovered by Kornberg in 1956. All the DNA
polymerase require the following:

1. A template DNA strand
   
   
2. A short primer (either RNA or DNA)
   
   
3. A free 3’ – OH in the primer.
   
   

They add one nucleotide at a time to the free 3’OH of the primer, and extend the primer
chain in 5’ → 3’ direction.

DNA polymerase is actually an aggregate of several different protein subunits, so it is
often called a holoenzyme. The holoenzyme also has proofreading activities, so that it
can make sure that it inserted the right base and nuclease (excision of nucleotides)
activities so that it can cut away any mistakes it might have made.

DNA polymerase I :

This enzyme was first purified by Kornberg in 1956. Hence, it is also called Kornberg
enzyme. This enzyme has three activities, which appear to be located in different
parts of the molecule.

A polymerase activity, which catalyses chain growth in the 5’ → 3’ direction

A 3’ → 5’ exonuclease activity, which removes mismatched base (DNA proof reading)

A 5’ → 3’ exonuclear activity, which degrades double stranded DNA (excision repair).

A exonuclease digests nucleic acids from one end (it does not cut DNA internally).

DNA polymerase II:

This enzyme repairs the damaged DNA. It has 5’ → 3’ polymerase and
3’→ 5’ exonuclease activities.

DNA polymerase III:

This enzyme is responsible for DNA replication in vivo. It has 5’ → 3’
polymerase and 3’ → 5’ exonuclease activities. DNA polymerase III is
a complex enzyme containing seven different polypeptides, and
5'to 3'of these polypeptides must be present for proper replicative

function. The 5' to 3' polymerase activity and the 5'to 3'exonuclease
activity are both present on the α polypeptide of DNA polymerase III.

The 3'to 5'proofreading activity of polymerase III is present on the
ϵ polypeptide.

Primase:

Is a part of an aggregate of proteins called the primeosome. The primase
(RNA polymerase) added to other proteins (forming a Primesome) makes
short pieces of RNA (RNA primers) that are recongnised by DNA polymerase III
to initiate replication.

As DNA polymerase III exhibits outstanding proofreading capabilities, which
prevent it to initiate a polynucleotide strand synthesis. Therefore it requires
a primer, a short piece of RNA (RNA primer) that it can recognize and elongate.
This RNA primer is eventually removed by RNase  and the gap is

filled in by DNA polymerase I.

Ligase:

Catalyze the formation of a phosphodiester bond given an unattached but
adjacent 3’OH and 5’ phosphate. This can fill in the unattached gap left
when the RNA primer is removed and filled in. The DNA polymerase can
organize the bond on the 5’end of the primer, but ligase is needed to

make the bond on the 3’ end.

The SSB  proteins (Single Strands Binding) stabilize the single strands thus
preventing them to zip back together and to form hairpin loops.
Single – stranded binding proteins are important to maintain the stability
of the replication fork. Single-stranded DNA is very labile, or unstable, so
these proteins bind to it while it remains single stranded and keep it from
being degraded.

Mechanism of DNA Replication

DNA synthesis is semiconservative (i.e., one of the parental DNA strands is conserved).

DNA replication begins at certain unique and fixed points called ‘Origin’ (ori)

Two enzymes DNA gyrase and DNA helicase, bind to the origin points and induce

the unwinding and separation of complementary strands of DNA double helix.

This separation is known as DNA melting.

Unwinding of DNA produces Y-shaped replication forks.

DNA polymerases proceed only in the 5’ to 3’ direction, DNA polymerase

require for their unction the presence of a :

i. Template DNA

ii. A primer (RNA or DNA, but only RNA is used in vivo).DNA polymerases can only

add nucleotides to the 3’OH group of a pre- existent primer

iii. Four deoxynucleoside triphosphates (dATP, dTTP, dGTP and dCTP), which are the

building blocks of DNA

As the two strands separate, the bases are exposed and RNA polymerase or

primase initiates transcription of the strand (3' to 5') and generates a 10 -60

nucleotide long RNA primer in 5' to 3' direction.

The free 3'- OH of this RNA primer provides the initiation point for the synthesis

of new DNA strand. DNA polymerase III adds deoxyribonucleotides to the 3'- OH group

of the last ribonucleotide of the RNA primer.

DNA polymerase I catalyzes the removal of the RNA primers by the concerted action

of its 5'to 3' exonuclease activity and its 5'to 3' polymerase activity.

DNA poly III progressively adds deoxyribonucleotides to the free 3'- OH of this growing

polynucleotide chain according to the base pairing rules (A=T; G=C). Consequently,

the replication of 3' to 5' strand of a DNA molecule proceeds continuously.

Because both strands of a DNA molecule are antiparallel, i.e., run in opposite directions,

this creates a problem. When the two strands unwind at the replication fork the leading

strands faces the DNA polymerase in correct 5’ to 3’ direction, so that the synthesis

of a long continuous complementary strand takes place.

On the lagging strand this is not possible; therefore, replication proceeds in a

discontinuous way, synthesizing short segments of DNA (always in the 5’→3’ direction)

called the okazaki fragments.

These segments are then joined together by the action of a DNA ligase. This short

discontinuous DNA segments were discovered by T. Okasaki, who exposed bacteria

to ³H -thymidine for a few seconds and found that fragments of DNA 1000 - 2000

nucleotides long. These fragments were called Okasaki fragemnts after their discoverer.

These are only 200 nucleotides long in eukaryotes.

DNA polymerases can only elongate a primer molecule; they cannot start a new chain

by themselves. This poses a problem when initiating each Okasaki fragment,

which is solved by synthesizing a short segment of RNA that acts as the primer.

Each Okasaki fragment starts with an RNA segment. RNA is used (instead of DNA)

because it immediately provides a ‘tag’ indicating that this part of the molecule has

not yet been subjected to the proofreading mechanism and must be replaced.

This RNA segment is subsequently removed by repair enzymes, the gap is filled

by DNA polymerase, and then joined to the neighbouring Okasaki fragment by a

DNA ligase to form a long polynucleotide chain.

The rate of movement of a replication fork in E.coli is about 10⁵  base pairs

per minute. In eukaryotes the polymerases are much less active and the rate

ranges from 500 to 5000 base pairs per minute.

In E.coli, the termination is signalled by specific sequences called ter elements. They

serve as binding site for protein Tus. The Tus protein binds to ter element and stops

helicase enzymes from unwinding DNA. This stops the movement of the replication fork.