The complimentary ends of the DNAs specifically associate under annealing conditions and are covalently joined through the action of an enzyme named DNA ligase. The enzyme produced by bacteriophage T4.
Mechanism of DNA ligase activity:
The E.Coli and T4 ligases share the property of sealing nicks that have 3'’-OH and 5’- P termini. Both enzymes under take a two step reaction, involving an ‘enzyme-AMP complex’.
- The E.Coli and T4 enzyme use different cofactors.
- The E.Coli enzymes uses NAD as a cofactor, T4 enzyme uses ATP.
The AMP of the enzyme complex becomes attached to the 5’-Phosphate of the nick; and then a phosphodiester bond is formed with the 3’-OH terminus of the nick, releasing the enzyme and the AMP.
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