DNA Polymerases – Kornberg (1957) isolated an enzyme from E. coli which had the ability to polymerise the nucleotides dATP, dTTP, dCTP and dGTP to form a polynucleotide in the presence of a primer and DNA template.
This enzyme was capable of synthesizing new DNA strands by proceeding in 5 ‘->3’ direction. Subsequent works of DeLucia and Carirns (1969), Knippers (1970) and Gefter et al (1969) showed that in prokaryotic cells there are at least three DNA polymerases – poly I, pol II, and poly III. Romberg’s DNA polymerase is equivalent to Pol I.
Polymerase I – This enzyme does not seem to be necessary as bacterial mutations lacking this are able to synthesize DNA. Poly I seem to be taking part in DNA repair.
Image Source: pnas.org
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Structurally poll is a single polypeptide chain with a molecular weight of 1, 09,000. Electron microscopic studies of the enzyme have shown that the enzyme has a number of functional sites –
(i) A template site for binding template DNA
(ii) Primer site for binding primer RNA segment
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(iii) A primer terminus site for 3′ – hydroxyl terminus of primer
(iv) A triphosphate site – locus for incoming deoxy ribonucleotide 5′ group
(v) Exonuclease site for 5 ‘—>3′ exonuclease activity.
Polymerase II – This is a single polypeptide chain with a molecular weight of 90,000, E.coli cell has about 40 molecules ofpol III. It helps in 5′-»3′ polymerization.
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Polymerase III – Discovered by Komberg and Gefter (1972) it is the most active among all the three enzymes. The enzyme is chiefly responsible for DNA chain elongation. It has a molecular weight of 5, 5000. The enzyme also acts as 5’—>3′ and3′-»5′ exonuclease..
DNA ligase. Also known as polynucleotide ligase, this enzyme joins the two fragments by catalysing the synthesis of a phosphodiester bond between a 3′- OH group at the end of one chain and 5’—>3’ – OH group at the other end of the other chain. The enzyme is necessary to join Okazaki fragments which are produced after discontinuous replication.
The enzyme has been isolated from E.coli. It is made up of a single polypeptide chain and has a molecular weight of 70,000. Each cell of E.coli is known to posses 2000-4000 molecules of this enzyme. DNA ligase has the following functions.
1. Joins Okazaki fragments during DNA replication
2. Helps in sealing single strand nicks in DNA duplex
3. Joins free ends of DNA (linear molecule) to form a circular chromosome.
4. Joins the segments of DNA during recombination during meiosis.