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.  Rate of replication in eukaryotes is about 10 - 100 nucleotides per
second.  To complete replication of simple eukaryotic genome 6 - 8 hours required.  In
prokaryotic circular DNA only one replication fork is present but in eukaryotic DNA several
replication forks are present.  Space between two-replication forks in eukaryotes is about 20kbps
apart.

THE REPLICATION OF CIRCULAR DNA IN E.COLI (Prokaryotic duplex

DNA replication) ( REPLICATION):

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

I) Initiation

            The replication begins at a specific initiation point called OriC 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 OriC site is situated at 74" minute near the ilv gene.  The OriC 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).

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

a) Dna A protein recognizes and binds up to four 9bp repeats in OriC to form a complex of
negatively supercoiled OriC 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) Dna A protein subunits then successively melt three tandemly repeated 13bp segments in the
presence of ATP at >=22*C (open complex).

c) 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.
d) 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.

e) 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.

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.

a) Initiation of synthesis and Elongation on the 5'-->3' template (synthesis of leading

strand) (If replication fork moves in 3'-->5' direction)

            The DNA daughter strand that is synthesized continuously on 5'-->3' template is called
leading strand.  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.
b) Initiation of synthesis and Elongation on 3'-->5' template when fork moves in 3'-->5'
direction (Synthesis of lagging strand) 

            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.
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.
III) TERMINATION: