DNA Replication in Prokaryotes:-
- Replication is the process of synthesis of DNA from the template DNA stranded or parental enzymes and protein factors.
- The replication of DNA is carried out during s-phase of interphase and all the proteins and enzymes required for DNA replication are synthesized during G1 phase.
- This process is also Klas DNA dependent DNA synthesis is.
- Prokaryotic DNA replication comprises of 3 steps.
Initiation of DNA Replication:-
- Initiation of DNA replication in prokaryotes is accomplished by the recognization of the origin, opening of the duplex to generate a region of single stranded DNA and capture of the DNA B (helicase) protein.
- Recognition of the origin occurs by the binding of DNA A protein.
Origin of replication in E. Coli is composed of 245bp which consists of 9mer and 13mer sites.
- DNA A- ATP complex (10-20 monomers of DNA A) bind to 9bp inverted repeat regions of oric and promotes opening of DNA in a region of 3directs repeats of a 13bp sequence.
- The opening occurs from right 13mers left word and requires -vely super coiled DNA along with HU or IHH (Interstitial Histone factor proteins).
- On DNA B or helicase is then trans fered along with DNA C to the site where DNA A is attached to the DNA.
- The function of DNA C is to tranfer the DNA B to DNA A site and it requires catalysis of ATP. Soon after the transfer of DNA B. DNA C is dissociated from DNA B.
- DNA B couses unwinding of DNA or addition of ATP, SSB, Mg2+ and gyrase. There is requirement of one SSB for one strand and two helicases are required for both strand (1helicase per one strand).
- Unwinding is followed by synthesis of RNA primer by a complex called primosome (primaset DNA B/helicase) and the primers are elongated by DNA polymerase lll hobenzyme.
- The elongated process is carried out by a mobile complex Klas replisome (DNA B + Primase + polymerase) SSB protein helps for maintaining the stability of single stranded DNA where as gyrase helps for prevention of super coiling formation.
Elongation Of DNA Replication:-
- Before the starting of elongation process the preinitiation and initiation complex is formed.
- This complex involves the components of DNA polymerase.
- Holoenzyme and this initiation complex is formed in two stages.
- Requirements for enlogation process are DNA is protein, primosome and DNA polymerase lll holoenzyme.
- DNA B protein unwinding the DNA from 5′ to 3′ direction and direction of unwinding requires DNA B to be placed on lagging strand ahead of the polymerase of a leading strand.
- The primosome synthesize primer of 100 nucleotide long RNA.
- DNA polymerase lll holoenzyme is a large multiprotein. Complex with at least 10subunits.
- The holoenzyme consists of a core polymerase and it’s accessory factors.
Replication for movement:-
- During initiation of elongation DNA B generates replication fork by opening the DNA the lagging strand template.
- DNA primase associate with DNA B and synthesize multiple primer for lagging strand. The dimeric polymerase lll holoenzyme ineract simultaneously with both template strand.
- For synthesis of lagging strand DNA polymerase act on the same direction while it acts in opposite directions in leading strand.
Continuous synthesis on leading strand:-
- The DNA polymerase lll holoenzyme complex on leading strand is extremely processive.
- After the attachment of RNA primer on the leading strand, DNA pol. lll elongated the chain by adding one after another nucleotide in continuous manner.
- The elongation proceeds in 5′ -3′ direction. Where the nucleotides are attached to the 3′ end of the RNA primer but these added nucleotides are complementary to the leading template strand.
Discontinuous Synthesis on lagging strand:-
- On lagging strand, DNA polymerase enzymes is less processive.
- The primase is activated by DNA B to synthesis primer of 10-20 nucleotide long on the lagging strand and DNA pol. lll holoenzyme interacts with primase and provides the signal from primase to dissociate from the fork.
- The primers are recognized by the polymerase on lagging strand and are utilised for synthesis of short fragments that is 1000-2000 nucleotide long called as okazaki fragments. After completion of okazaki fragments synthesis the core enzymes rapidly dissociate and rapidly associated with new B2 clamp to form rapidly associated with new B2 clamp to form another primer and it’s elongator.
For lagging strand, the attachment of and it’s elongation occurs in a loop like structure.
- Other proteins required for DNA elongation are:-
(a) DNA gyrase which removes topological strain associated, with DNA unwinding.
(b) SSB proteins for stabilizing unwound DNA.
(c) DNA pol.l for removal of RNA primer DNA pol.l for filling the gap due to removal of RNA primer and DNA ligase which converts okazaki fragments into a continuous DNA strand and also fills the gap between nucleotide by forming 3′-5′ phosphodiester bond.
- In prokaryotic chromosome, there is a specific sequence called ter site where TBP (Ter Binding Proteins) or TUS proteins binds.
- In E. Coli there are 3 ter sites for counter clockwise fork and 3 tersites for clockwise fork.
- The TUS ter complexes formed at ter sites stops the replication fork by inhibiting the helicase enzymes.
- Due to the moulting of replication fork, the helicase can’t proceed further and the replication fork moves from left to right and the replication is completed.
Each parental strand with their corresponding new DNA strands get separated from each other and this is Klas the semi conservative mode of replication.