Replication in Prokaryotic cell

1. Compiled by
V. Magendira Mani
Assistant Professor,
PG & Research Department of Biochemistry,
Islamiah College (Autonomous),
Vaniyambadi,
Vellore District – 6357512,
Tamilnadu, India.
magendiramani@rediffmail.com ; vinayagam magendiramani@academia.edu

2. REPLICATION IN PROKARYOTES
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.
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

3. 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) It is the
point of DNA open up and form open complex leading
to the formation of prepriming complex to initiate
replication process.
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 basepair sequences each (TTATCCACA).

4. The sequence of reactions in the initiation process is as
follows:
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.
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.

6. 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.

8. 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.
REPLISOME:
It is the multiprotein structure that assembles at the bacterial
replicating fork to undertake synthesis of DNA. It contains
DNA polymerase and other enzymes.

9. 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.
Leading strand synthesis begins with the synthesis of RNA
primer (10 -60 nucleotides) by primase (DNA g protein)

10.  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.

11. Synthesis of lagging strand
Initiation of synthesis and Elongation on 3'-->5' template
when fork moves in 3'-->5' direction
The daughter DNA strand which is synthesized in
discontinuous complex fashion on the 3'-->5' template is
called lagging strand. It occurs in the following steps:
i) Synthesis of Okazaki fragment:
To the RNA primer synthesized by primosome, 1000-2000
nucleotides are added by DNA pol-III to synthesis Okazaki
fragments.
ii) Excision of RNA primer:
When the Okazaki fragment synthesis was completed up
to RNA primer, then RNA primer was removed by DNA
pol - I using its 5'-->3' exonuclease activity.

12. iii) Filling the gap (Nick translation)
The gap created by the removal of primer, is filled up by
DNA pol - I using the 3'-OH of nearby Okazaki fragment
by its polymerizing activity.
iv) Joining of Okazaki fragment: (Nick sealing)
Finally, the nick existing between the fragments are sealed
by DNA ligase which catalyze the formation of
phosphodiester bond between a 3'-OH at the end of one
strand and a 5' - phosphate at the other end of another
fragment. The enzyme requires NAD for during this
reaction.

14. 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.
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.

15. REGULATION OF PROKARYOTIC REPLICATION
Especially initiation of replication is regulated. Dna A protein
when available in high concentration then ratio of DNA to cell
mass is quiet high but at low Dna A concentration, the ratio
found to be low. This shows that Dna A protein regulates the
initiation of replication.
The sequence most commonly methylated in E.Coli is GATC
including in three of 13mer sequence. Thus, the observation that
E.Coli defective in the GATC methylation enzyme are very
inefficiently replicated, suggests that the DNA replication trigger
also responds to the level of Ori C methylation.

16. ALL THE BEST
By
VMM
V. Magendira Mani
Assistant Professor,
PG & Research Department of Biochemistry,
Islamiah College (Autonomous),
Vaniyambadi,
Vellore District – 6357512,
Tamilnadu, India.
magendiramani@rediffmail.com ;
vinayagam magendiramani@academia.edu
https://tvuni.academia.edu/mvinayagam