Replication is a means to produce new molecules that have the same base sequence.

It occurs during interphase of the cell cycle.

DNA replication is semi conservative in nature as proved by Meselson-Stahl experiment.

Meselson-Stahl experiment(1957) Bacteria were grown in a medium containing nitrogen

15(N15) for several generations.

If the medium contains no other nitrogen source, the E.coli will use N15 and incorporate it into their DNA.

Eventually it will have only N15

Once the E.coli had only N15 they were passed into a growing medium containing only N14

N15 is heavier than N14 making new incorporation of nitrogen making easy to distinguish

The differences were measured according to the densities of the new strands.

DNA Replication is semi conservative

Replication of duplex DNA involves a conglomerate of enzyme activities.

Initiation of synthesis of a DNA strand is

accomplished by a protein complex called the

primosome, which consists of DnaB helicase

and DnaG primase.

Elongation is undertaken by another complex of

proteins called replisome. As replisome moves

along DNA,the parental strands unwind and

daughter strands are synthesised.

At the end of replicon, termination reactions are

necessary

Proteins of the E.colireplisomePROTEINS FUNCTIONS

1) SSB

2) DnaB protein(Helicase)

3) Primase (DnaG protein)

4) DNA Pol lll

5) DNA Pol l

6) DNA ligase

7) DNA gyrase(DNA

Topoisomerase ll)

1) Binding to ss DNA

2) DNA unwinding; Primosome constituent

3) RNA primer synthesis; Primosome constituent

4) New strand elongation

5) Filling of gaps; excision of primers

6) Ligation

7) Supercoiling

Features of E.coli replication origin,ori c.

•Five repeats of a 9bp sequence (R sites) that are binding sites for protein Dna A.

•A=T rich region called the DNA unwinding element (DUE).

•Three additional Dna A binding sites (I sites), binding sites for proteins IHF,FIS.

Proteins (in addition to proteins of replisome ) required to initiate replication at E.coli origin, ori c

Proteins Functions

Dna A protein

DnaC protein

HU

IHF

FIS

Dam methylase

Recognizes ori sequence; opens duplex at specific sites in origin

Required for DnaB binding at origin

Histone like protein; stimulates initiation

DNA binding protein; stimulates initiation

DNA binding protein;stimulates initiation

Methylates (5’)GATC sequence at ori c

Steps of initiating DNA replicationHelicase unwinds DNA at the replication

fork, hence DNA is forced to rotate.

Primase then synthesises short stretches of RNA primer.

Due to unwinding of DNA duplex, twists are created in the DNA ahead and hence tension builds up.

DNA gyrase relieves this tension by adding negative supercoils in DNA helix.

SSB proteins binds to the bare single stranded DNA.

Clamp proteins form a sliding clamp around DNA

Figure showing DNA Replication.

Elongation

As the replication fork advances, daughter strands are synthesised on both of the exposed parental single strands.

Elongation On the leading strand DNA synthesis can proceed

continuosly in the 5’ to 3’ direction.

On the lagging strand a stretch of parental DNA must be

exposed, and a segment is synthesised in the reverse

direction (opposite to fork movement).

A series these fragments (okazaki fragments) are

synthesised each 5’ to 3’; then they are joined by ligase

to create an intact lagging strand.

Hence this mode of replication is called semi

discontinuous replication

Figure showing Okazaki fragments.

Nick translationIn this process, an RNA or DNA strand

paired to a DNA is simultaneously

degraded by 5’ to 3’ exonucleolytic activity

of DNA Pol l and replaced by the

polymerase activity of the same enzyme.

Nick translation is done for removing the

RNA primers in front of the okazaki

fragment.

ProcessivityIt is the average number of

nucleotides added to a growing DNA

strand before the polymerase

dissociates. Processivity of polIII is

much higher than that of polI.

Inhibitors of DNA ReplicationActinomycin binds to DNA duplexes, hence

inhibits replication.

Mitomycin C is a potent DNA cross linker.

Plicamycin is an RNA synthesis inhibitor.

Quinolones act upon DNA gyrase as a

topoisomerase inhibitor.