The mechanism of DNA replication in is as follows –
1. Stage of replication during cell division:
It has been observed that replication takes place during inter phase between two mitotic cycles. During inter phase the amount of DNA doubles due to synthesis. However, DNA synthesis does not take place during the entire inter phase. It is confined to the S phase.
2. DNA synthesizing genes:
In E.coil the overall process of replication requires the products of the genes dnaA, dnaB,dnaC-D dnaE and dnaG.
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The gene products of A, C and D are required for the initiation of fresh
Replication and the products of E and G are required for the fork movement for replication.
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The product of G appears to be necessary for initiating the synthesis of DNA fragments. The E.coli also possesses a gene for the origin of DNA synthesis which is about 74 minutes from the beginning.
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There is also a gene for the termination of the replication. There are additional genes to control the synthesis of the three DNA polymerase enzymes. PolA and polB regulate the synthesis of polymerase I and polymerase II enzymes while G and E regulates polymerase III enzyme.
3. Initiation of replication with a nick:
Unlike in the case of eukaryotes when the molecules are liner in prokaryotes the DNA molecules are circular. As such there has to be break in the circle to begin replication.
The DNA consists of many replicating units or replicons. According to one hypothesis replication is initiated only after a break or nick in one of the two parental strands. The cut is presumed to take place in the helix at a specific initiation point. This cut or incision is made by a specific endonuclease enzyme.
4. Origin of replication:
Replication does not begin randomly it always begins with a specific site called ori site and is regulate by a gene and its product. It has been discovered that in E.coli the origin of replicant point as about 74 minutes on the map.
5. Role of plasma membrane:
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There is evidence to indicate that E.coli consists of an enzyme complex at the point of attachment of DNA to the membrane this point of attachment is known as the replicating point.
The DNA thread moves through the replicating point and duplicates itself in the process. In this process the replication is exactly opposite of what is seen in eukaryotes where it is the enzyme that moves along the strand.
The exact relationship between the plasma membrane and the DNA is not known. Quite probably the membrane lipids may have some role to play in the process of initiation. Polymerase II and polymerase III enzymes are seen at the point of attachment.
6. Strand unwinding:
The DNA helix has to first unwind in order to separate the two strands for beginning the replication. A study of the unwinding process in T2 bacteriophages shows that each DNA molecule has to undergo 20,000 rotations in unwinding during replication.
As the molecule itself consists of 2 lakh nucleotide pairs, in these phages replication must go on simultaneously along with unwinding.
The unwinding of the helix is controlled by enzyme proteins commonly referred to as DNA unwinding proteins. These proteins selectively bind to the single strands of DNA and promote unwinding of the coil.
The region of the double helix where the proteins bind forms a Y shaped structure called the replication fork. It has been found out that nearly 200 molecules of the proteins are found at each fork.
Chemical analysis of the DNA unwinding protein shows that each molecule is a tetramer having 4 sub units. Experimental evidence in E.coli has shown that the addition of DNA unwinding proteins to the medium increases the phase of the unwinding of the DNA molecule.
7. Role of Swiveling Protein:
As the replication fork moves downwards along the parental strands, unwinding begins. For every ten nucleotides there will be one rotation of unwinding.
As the original configuration of the molecules is in the form of a wound helix in circular DNA molecules present in prokaryotes untwisting or unwinding imposes a strain.
To some extent this strain can be relieved by additional twisting called super twisting in the un-replicated part of the DNA.
However this super twisting has a limit and ultimately even the super twisting should be unwound for the replication fork to continue.
The relieving of the strain imposed by unwinding in brought about by proteins called super helix relaxing proteins.
These proteins introduce small cuts in the non replicating region of DNA thereby making one strand to rotate upon the other; thus relieving the strain. The breaks can then be healed by ligation.
8. DNA Template:
To synthesis a fresh strand of DNA apart from the enzymes the most important requirement are the nucleotides and a DNA template.
The 4 nucleotides are adenine, guanine, cytosine and thymine. The previously existing DNA can serve as a template on which can be assembled fresh nucleotides.
The template DNA can be either single stranded or double stranded. Single stranded DNA is found naturally in some bacteriophages template DNA acts as a mould on which new strand of DNA is synthesized by complementary base pairing.
9. RNA Primer:
DNA synthesis cannot be initiated on a DNA primer. DNA polymerase enzymes can initiate DNA synthesis only if there is a RNA primer. In other words synthesis of a fresh strand can begin only if there is a small piece of a strand already.
This small piece which is RNA is called RNA primer. The RNA primer is a short polynucleotide chain synthesized by the DNA template close to the point of replication.
In all probability this is catalyzed by a modified RNA polymerase enzyme. It has been noticed that in E.coli RNA polymerase is necessary to begin the replication.
10. Elongation of chain:
Synthesis of new DNA strand takes place by the addition of fresh nucleotides to the 3′-OH group of the last nucleotide in the RNA primer. This synthesis takes place in 5′->3′ direction and the enzyme those catalyses this is DNA polymerase III.
The newly synthesized DNA chain has an RNA primer attached to its 5′ end. After the synthesis of the chain, the RNA primer is hydrolyzed by the exo nuclease activity of DNA polymerase I.
The resulting gap left by the RNA primer is filled by DNA nucleotides by the catalytic activity of DNA polymerase I. The freshly formed nucleotide chain is joined to the existing chain by the lygase enzyme.
Several opinions have been put forward to account for the presence of an RNA primer to initiate the synthesis of DNA chain. According to one opinion if any errors occur during the initial synthesis it can be eliminated when the RNA primer is removed.
11. Direction of Replication:
It has been seen that in prokaryotes the replication may proceed in one or both directions from the point of origin. Thus replication may be unidirectional or bidirectional.
Bidirectional replication has been seen in E.coli, Bacillus subtilis, Salmolella, typhimirum, E.coli phages etc. Unidirectional replication has been reported bacteriophages P2 and 186 and also in the mit DNA of mouse LD cells. Bidirectional replication has an advantage in that the origin is completely duplicated before the complication of replication.
12. Formation of Replicating Forks:
At the point where the two strands get separated to initiate replication a replicative fork is found. The shape of the replicating fork is in the form of the English alphabet Y.
When bidirectional replication takes place, the separated strands between the two forks appear like the bubble or a convex lens under the electronic microscope. The speed of movement of the two forks need not be the same.
In organisms like E.coli where the chromosome is circular there will be only two replicating forks, where as in eukaryotes there could be several thousand replicating forks. This large number of forks is due to the fact that replication begins simultaneously at many points along the length of DNA.
Occasionally however even in bacteria when replication is going on at one point another replication may start at another point this will facilitate in completing the replication in shorter period.
13. Discontinuous Replication (Okazaki fragments)
The duplication of the strand of DNA takes place by the movement of replication forks. Replication of the two parental strands will take place at the same time as the fork moves along. However all the DNA polymerase enzymes can enhance the length of the strands only in the 5′-»3′ direction.
The strands of DNA on the other hand polarize in opposite directions. This naturally possesses a question as to how can DNA polymerase bring about simultaneous replication.
While the enzyme moves forward on one DNA strand, simultaneously it cannot move backward on the other. Okazaki et al (1968) have provided an answer to the above problem.
According to them only one strand 3 ‘->5′ is continuously replicated while the other 5’—>3’ replicates in a discontinuous manner and synthesize only short fragments of DNA. These short fragments have been called Okazaki fragments.
The direction of synthesis of Okazaki fragments in opposite to the movement of the replicating fork. Eventually Okazaki fragments are joined together by the enzyme polynucleotide ligase. In prokaryotes the Okazaki fragments are as long as 1,000- 7,000 nucleotides.
According to another suggestion (Okazaki et al) both the strands may replicate discontinuously and the fragments formed may join up later. Evidence obtained form in vitro culture of E.coli has shown that both the strands are synthesized discontinuously.