Mechanism of DNA Replication
DNA replication involves four important stages. These are
- Initiation of DNA replication
- Unwinding of DNA helix
- Formation of Primer strand
- Elongation of new strand
1.Initiation of DNA replication:-
Replication of DNA always begins at a definite site called replication origin. In virus and bacteria there is only one such replication origin present. e.g E-coli.But in eukaryotes there are several thousands of replication origins are present. These linearly arranged units are known as replicons.e.g-
Organisms No. of replicons
Yeast 500
Drosophila 3500
Toad 1500
Mouse 25000
Bean 35000
In Escherichia coli replication is due to multienzyme complex known as replication apparatus or repliosome.
2.Unwinding of DNA helix:-
Now unwinding occurs in the parental DNA molecule in such a manner that its internal bases are exposed to the replication enzymes.Unwinding occurs due to a specific enzyme called Helicase or rep protein. This leads to formation of super coils which are then removed by the action of another enzyme called DNA gyrase or Topoisomerase.
Unwinding of DNA molecule separates the two strands to form a Y-shaped structure called replication fork.Then the exposed single strands ae stabilised by a protein called single stranded binding protein (SSB) or the helix destabilizing protein. The SSB proteins holds open the two separated strands to form replication fork.
3.Formation of Primer strand:-
After that opposite to each of the parental strand a new strand is synthesized. The parental strand acts as a template strand. The enzyme which is meant for replication is DNA Polymerase. In prokaryotes it is of three types i.e DP-I ,DP-II and DP-III. Out of which DP-I and DP-II are concerned with DNA repair where DP-III takes part in actual DNA replication .But in eukaryotes DNA polymerase is of five types i.e- DP-𝞪, DP-𝜷 ,DP-𝛄, DP-𝞭 and DP-Ɛ. Out of which DP-Ɛ takes part in DNA replication.However in both the cases DNA polymerase is incapable of initiating DNA synthesis. It is unable to deposit the first nucleotide in a daughter strand. For that reason another enzyme primase is initiating the synthesis by producing a short primer strand of RNA . Some other enzymes are also involved in this process and are collectively known as primasomes.
This primer strand then elongates and removed enzymatically once the initiation of DNA synthesis is completed. In E.coli DP-I helps in removal of RNA primer. In eukaryotes DP-𝛂 synthesizes primer strand.
4.Elongation of new strand:-
DNA replication takes place in 5'-3' direction. It occurs after the formation of primer strand. At this time deoxyribonucleotides are added only to the 3'OH end of the new strand. These are ATP,GTP,CTP and TTP. In each of the addition a molecule of pyrophosphate is simultaneously released. Thus the nucleotide at 3' carbon of sugar is always the most recently added nucleotide to the chain.
As proceeds of the two parental strands synthesis of daughter or new strand occurs continuously along the upper strand in 5'-3'OH direction initially known as leading strand. Similarly synthesis of another daughter strand along the lower parental strand in 3'OH-5'P direction occurs in the form of short pieces known as lagging strand. These pieces are called as okasaki fragments. Those are 1000 to 2000 in number (nucleotide pieces). Later on they joined together by the enzyme DNA ligase to form a continuous daughter strand.
So at the end two DNA molecules are formed from one molecule where each of the daughter DNA is made of one parental or old strand and other new or complementary strand.
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