Prokaryotic Replication

Enzymes of DNA replication:
The process of DNA replication involves many enzymes, which work in one accord for the duplication process of DNA. Those enzymes are discussed below one by one
 
 (i) DNA gyrases:
These enzymes are also called topoisomerases because they relieve the topological stress caused the unwinding of the double stranded DNA. They introduce negative supercoils from the other end of replication fork to relieve the stress. These are again classified into two groups namely topoisomerases type I and topoisomerases type II based on their mode of action. Topoisomerase I cause a break or nick on only single strand of the DNA on the other end of the replication fork and rotate freely in order to relieve topological stress. Topoisomerase II also acts in a slightly different manner by forming a break on both the strands of DNA.

(ii) DNA polymerase I
Sir Arthur Kornberg discovered and isolated this enzyme in 1960’s and suggested that it is involved in DNA replication. Thus it is also known as Kornberg enzyme. It is a single polypeptide chain and 103 Kilo Dalton enzyme. This enzyme on hydrolysis results in a larger fragment and a smaller fragment. The larger fragment is also known as klenow fragment, which is of 67 Kilo Dalton and the smaller fragment is about 37 Kilo Dalton.

This enzyme is popularly known as an editing or repair or proof reading enzyme because it consists of 5’ to 3’ polymerization, 5’ to 3’ exonuclease and 3’ to 5’ exonuclease activities, which help in repairing a synthesized strand. The other functions such as nick translation and strand displacement are also some properties of this enzyme.

(iii) DNA polymerase II
This enzyme is similar to the DNA polymerase I and it is thought to be a repairing enzyme too. The function and working of this enzyme is yet to be eludicated.
 
(iv) DNA polymerase III
This is a very essential enzyme in the process of replication for DNA polymerization at high speeds. Its processivity is 1000 nucleotides for second. It is a multimeric or a holoenzyme and a 900 Kilo Dalton enzyme with about 10 different subunits. All the ten subunits have ten different functions and the subunits are namely Alpha (), Epsilon (), Theta (), Beta (), Delta (), Delta dash (), tau (), chi (), psi (), and Gama ().

For instance subunit has 3’ to 5’ exonuclease activity which is a proof reading or editing in function and subunits together are termed as core enzyme. The other subunits increase the processivity of the enzyme.

(v) DNA - A protein
It is also known as initiator protein since its function is to identify or recognize the ori ‘C’ site and initiates the process of replication.

(vi) DNA - B protein
This is popularly known as the enzyme Helicase. The main function of this enzyme is to unwind helices of the double stranded DNA by breaking the hydrogen bonds between the strands and separate the strands. Hence the helicase is given to the enzyme.

(vii) DNA - C protein
It is a single polypeptide chain helper enzyme to the DNA – B protein that helps in binding of the DNA – B protein or the helicase to the double stranded DNA for unwinding the DNA.

(viii) SSBPs
The SSBPs is in fact an acronym that stands for single strand binding proteins, which are otherwise called as helix stabilizing proteins. It is a 74 Kilo Dalton enzyme that binds to the open single strands produced by the helicase enzyme and prevents the reunion of the strands.

(ix) Primase
Short RNA strands are called primers and the enzyme that synthesizes these primers is DNA primase. They are very essential in the process of replication because these short RNA oligonucleotides bind to the DNA strand in the initiation stages of replication and kick starts the replication process by providing free 3’ OH end to the polymerase enzyme. Once the polymerase completes synthesizing the new strand the primers are removed or excised and replaced with the DNA by the DNA repair enzymes.

(x) DNA ligase
DNA ligases are the enzymes that are used to seal or stick the DNA fragments together that are formed from endonuclease enzyme activity. They actually form a phosphodiester bonds between free 3’ OH end and free 5’ P end. They are of two types namely Ecoli DNA ligase and T4 DNA ligase, which can seal staggered ends and blunt ends respectively.