REPLICATION.

Replication is a process of duplication of DNA ,in which the

parental DNA is copied accurately to produce daughter DNA.
The daughter DNA (replica of the parental DNA) thus
produced are transferred to the daughter cells during cell
division.

Replication is semiconservative.

The process of replication in eukaryotes and prokaryotes can
be studied in 3 stages :
1.INITIATION.
2.ELONGATION.
3.TERMINATION.

I. Replication in prokaryotes :
Process is from studies made on bacterium,E.coli.
About 20 enzymes and proteins take part in prokaryotic
replication to overcome the structural complexity of DNA and
to achieve accuracy in copying.


INITIATION :
1.A specific protein dnaA locates the site of origin of replication
on parental strand. This site is usually a short sequence of A=T
base pairs. There is 1 site of origin in prokaryotic DNA.


2.Two other proteins dnaB & dnaC ,which are helicase enzymes
bind to the DNA strands and start unwinding them.
Helicases act as zip openers & are responsible for the separation
of strands as they move along the DNA helix.Helicases utilize
ATP for energy supply during the process.
This leads to the formation of a bubble like structure initially that
eventually forms REPLICATION FORK.


3.Separation of strands by helicases produces topological stress in
the supercoiled structure of DNA,which is relieved by
topoisomerases (also known as DNA gyrases) .
These enzymes cut the strands (nuclease activity) and reseal
(ligase activity) them.This mechanism relieves the strain over
the helical structure and make it reasdy for replication.

4.Further a group of proteins called single stranded DNA binding
(SSB) proteins bind to the single stranded DNA separated by
helicases &
prevent them from recoiling until the replication is completed in
that region.




5.The synthesis of new strand of DNA needs a pre-existing primer.
Primer which is present on the template strand is a short segment of
RNA synthesized earlier by an enzyme called primase ( a RNA
polymerase).
A template strand is the parental DNA strand which is copied.


Replication of DNA occurs in 5 to 3 direction,
simultaneously on both strands.
One strand of DNA (leading strand) is synthesized
continuously,
Whereas the opposite strand of DNA (lagging strand) is
synthesized in pieces ,called okazaki fragments ,in a
discontinuous manner.
A single primer occurs on the leading strand, multiple primers
exist on the lagging strand ( 1 / okazaki fragment).

At this stage replication is initiated by the enzyme DNA
polymerase III which adds a deoxyribonucleotide residue to the
RNA primer molecule.

The leading strand proceeds (5-3) in the same direction as that
of replication fork movement ,whereas the lagging strand runs
(5-3) opposite to the direction of replication fork movement.

ELONGATION
The synthesis of a new DNA strand by DNAP III occurs antiparallel
to the template strand as per the complementary base pairing and
proceeds in 5-3 direction.

The incoming deoxyribonucleotide residues are added one after
another to the 3 end of the growing DNA strand. A molecule of
pyrophosphate is released forming a phosphodiester linkage
between the two adjacent nucleotides.

leading strand synthesis proceeds continuously,uninterrupted as
the parent DNA is separated by helicases.
DNA synthesis proceeds rapidly & steadily on both the leading &
lagging strand templates at the same time,catalyzed by 2 sets
of DNAP III .

DNA synthesis on the lagging strand occurs as small pieces (in
5-3 direction), discontinuously (okazaki fragments)
Once the synthesis of an okazaki fragment is completed,
Replication stops & DNAP III dissociates & reassociates with a
new RNA primer to synthesize a new okazaki fragment.

Once an okazaki fragment is synthesized,its RNA primer is
removed & replaced with a DNA fragment by enzyme called
DNAP I.

The gap between the DNA synthesized by DNAP III & the small
fragment of DNA synthesized by DNAP I is bridged by a
phosphodiester linkage.
This nicksealing reaction is catalyzed by an enzyme called
DNA ligase ,which uses ATP for this purpose.

Apart from the main function of DNA synthesis,DNAP IIII also
has a proof reading activity.
It checks the incoming nucleotides & allows only the
complementary base pairing preventing any sort of
mismatching

TERMINATION:
The synthesis of DNA continues until it reaches a region known
as terminus sequence. once the replication fork enters into
Terminus region,it cannot make exit & replication comes to an
endin the circular chromosome of E.coli.Any DNA repair is
done by DNAP II.


REPLICATION IN EUKARYOTES :
Despite its structural complexity, the eukaryotic genome
replicates on similar lines as that of bacteria.
There are 5 distinct DNAP in eukaryotes :
1.DNAP alpha : synthesizes primers.
2.DNAP beta : similar to DNAP II DNA repair.
3.DNAP gamma : replicates mitochondrial DNA.
4.DNAP delta : analogous to DNAP III .
5.DNAP eta : DNAP I , removal of RNA primers of
okazaki fragments on lagging strand.
Replication in eukaryotic cells can also be studied under three
stages : Initiation , Elongation and Termination

INITIATION :
There are multiple sites of origin of replication at which the
replication is initiated.
The helicases bind to both the strands of parental DNA to
separate them.The strain thus produced is relieved by
topoisomerases (DNA gyrases).
A single stranded DNA binding protein called replication protein
a (RPA) binds to the separated parental strands & prevents
their reunification as helix.
This forms a perfect structure of replication fork.
Primase (RNAP) associates with DNAP alpha and synthesizes
primers .once the primer is formed,primase & DNAP dissocite
from the DNA.




2.ELONGATION :

A protein called replication factor C (RFC) binds to the primer.
RFC acts a clamp loader to another protein named proliferating
cell nuclear antigen (PCNA).
PCNA forms a circular clamp around DNA which is capable of
moving on it freely.
At this stage DNAP delta bids to the sliding clamp & starts
elongating the new DNA strand.The synthesis is continuous on
the leading strand, discontinuous on the lagging strand.

.
Once the synthesis of okazaki fragments is completed, the RNA
primer is removed by 2 enzymes RNase H and FENI (flap
endonuclease I) .
The gap formed is filled by DNAP eta. The small nick is sealed
by DNA ligase.

3.TERMINATION :
The termination of repication in eukaryotic cells involves special
structures called telomeres at the end of the linear
chromosome.
The ends of the chromosome are not replicated by DNAP.
Telomeres ,the specialized structures occuring at the ends of the
chromosomes are thousands of repeat sequences of 6
nucleotides (TTAGGG).


Telomerases are the enzymes that synthesize telomeres
Telomerase contains RNA apart from protein.
The RNA component of the enzyme acts as a template for the
synthesis of telomere (RNA dependent DNA synthesis).
The telomere synthesis is thus extended by telomerase so that the loss
of DNA at the ends of chromosomes is minimized during
replication.
There after the replication in eukaryotes is completed
producing 2 daughter DNA.
INHIBITORS OF REPLICATION

The differences between the replication in bacteria & human
cells give scope for developing antibiotics that target bacterial
replication but unaffects the human calls.

Several drugs ciprofloxacin, nalidixix acid ; act by inhibiting the
enzymes of bacterial replication.
Drugs targeting human replication enzymes such as
topoisomerases ,telomerases arrest new DNA synthesis & thus
cell division. They are effective in the treatment of cancer.
Eg: doxorubicin,adriamycin,etoposide.
Certain nucleotide analogues such as 6-mercaptopurine ,5FU
inhibit human DNAP. They are also used as anticancer drugs.
Thankyou.