a presentation on DNA replication in Prokaryotes. Office 2010 is highly recommended.

1. Presented by-
Utpal

2. THE STARTING POINT

3. Is life

4. The Central Dogma

5. REPLICATION
• Replication is an enzymatic process in which synthesis of
a daughter or progeny duplex DNA molecule, identical
to the parental duplex DNA occurs.
Rate of replication in E.Coli (prokaryotic cell) is 1500
nucleotides per second. To complete replication of
whole E.Coli genome it takes 40 minutes.

6. • The synthesis or replication of DNA molecule can
be divided into three stages
I) Initiation (Formation of Replisome)
II) Elongation (Initiation of synthesis and elongation)
III) Termination

9. I) INITIATION
• The replication begins at a specific initiation point
called Ori C point or replicon. (Replicon: It is a unit
of the genome in which DNA is replicated; it
contains an origin for initiation of replication).
• The Ori C site consists of 245 basepairs, of which
three of 13 basepair sequence are highly conserved
in many bacteria and forms the consensus
sequences (GATCTNTTNTTTT). Close to OriC site,
there are four of 9 base pair sequences each
(TTATCCACA).

10. a) Dna A protein recognizes and binds up to four 9 bp
repeats in Ori C to form a complex of negatively
supercoiled Ori C DNA wrapped around a central core of
Dna A protein monomers. This process requires the
presence of the histone like HU or 1 HC proteins to
facility DNA bending.

11. b) Once the four 9 bp repeats are occupied 20 – 40
additional Dna A monomers bind, so that entire Ori C region
is complexes with Dna A protein.
c) The resulting complex resembles a nucleosome with
negatively supercoiled Ori C DNA wrapped around a DNA
core.

12. d) HU, a histone like protein prevents nonspecific
initiation at sites other than the Ori C.
e) Dna A protein subunits then successively melt three
tandemly repeated 13 bp segments in the presence of
ATP, which results in the formation of 45 bp open
complex.
f) The Dna A protein then guides a Dna B - Dna C
complex into the melted region to form a so called
prepriming complex. The Dna C is subsequently
released. Dna B further unwinds open complex to form
prepriming complex.
g) DNA gyrase, single stranded binding protein (SSB),
Rep protein and Helicase - II are bound to prepriming
complex and now complex is called as priming
complex.

13. h) In the presence of gyrase and SSB, helicases further unwinds
the DNA in both directions so as to permit entry of primase and
RNA polymerase. Then RNA polymerase forms primer for leading
strand synthesis while primase in the form of primosome
synthesis primer for lagging strand synthesis.
f) To the above complex, DNA polymerase - III will bind and
forms replisome.

15. II) ELONGATION
• Now the stage is set for the initiation of synthesis
and the elongation to proceed. But this occurs in two
mechanistically different pathways in the 5` 3'
template strand and 3` 5` template strand.
• Initiation of synthesis and Elongation on the 5` 3'
template (If replication fork moves in 3` 5'
direction).
• Synthesis of leading strand
• The DNA daughter strand that is synthesized
continuously on 5` 3' template is called leading
strand.

16. • Leading strand synthesis begins with the
synthesis of RNA primer (10 -60 nucleotides)
by primase (DNA g protein).
• DNA pol-III synthesizes DNA by adding 5'-P of
deoxynucleotide to 3'-OH group of the already
presenting fragment.
• Thus chain grows in 5` 3' direction. The
reaction catalyzed by DNA pol-III is very fast.
The enzyme is much more active than DNA pol
- I and can add 9000 nucleotides per minute
at 37*C. The RNA primer that was initially
added by RNA polymerase is degraded by
RNase.

18. III) TERMINATION
• Termination occurs when the two replicating forks
meet each other on the opposite side of circular
E.Coli DNA. Termination sites like A, B, C, D, E and F
are found to present in DNA. Of these sites, Ter A
terminates the counter clockwise moving fork
while ter C terminates the clockwise moving forks.
The other sites are backup sites.
• Termination at these sites are possible because, at
these sites tus protein (Termination utilizing
substance) will bound to Dna B protein and inhibits
its helicase activity.

19. • And Dna B protein released and termination
result.
• After the complete synthesis, two duplex DNA
are found to be catenated (knotted). This
catenation removed by the action of
topoisomerase. Finally, from single parental
duplex DNA, two progeny duplex DNA
synthesized.