DNA replication is semi-conservative, as proved by the Meselson-Stahl experiment. It occurs during interphase and involves unwinding of the DNA double helix by helicase, followed by synthesis of new strands by DNA polymerases with RNA primers provided by primase. Replication proceeds bidirectionally from an origin of replication and results in two identical copies of the original DNA molecule.

2.  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.

3. 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.

5. DNA Replication is semi
conservative

6. 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

7. Proteins of the E.coli
replisome
PROTEINS FUNCTIONS
1) SSB 1) Binding to ss DNA
2) DnaB protein(Helicase) 2) DNA unwinding; Primosome
constituent
3) RNA primer synthesis; Primosome
3) Primase (DnaG protein) constituent
4) New strand elongation
4) DNA Pol lll 5) Filling of gaps; excision of
primers
5) DNA Pol l
6) Ligation
6) DNA ligase 7) Supercoiling
7) DNA gyrase(DNA

8. 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

9. Proteins (in addition to proteins of replisome )
required to initiate replication at E.coli origin, ori
c
Proteins Functions
 Dna A protein  Recognizes ori sequence;
opens duplex at specific sites in
origin
 DnaC protein  Required for DnaB binding at
origin
 Histone like protein; stimulates
 HU initiation
 DNA binding protein; stimulates
 IHF initiation
 FIS  DNA binding protein;stimulates
initiation
 Dam methylase  Methylates (5’)GATC sequence
at ori c

10. Steps of initiating DNA
replication
Helicase 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.

11. Figure showing DNA
Replication.

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

13. 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

14. Figure showing Okazaki
fragments.

15. Nick translation
In 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.

16. Processivity
It 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.

17. Inhibitors of DNA
Replication
Actinomycin 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.