Question

DNA ligase forms an activated intermediate in its mechanism. Explain, using words and/or diagrams as appropriate, what the activated intermediate is and why it favours the subsequent ligation of the 3′ and 5′ groups of the DNA

Answer 1

DNA ligase is a class of an enzyme ligases, which seals to two adjacent DNA fragments by forming the phosphodiester bond.

This this reaction is catalyzed by usage of energy ATP due to formation of phosphodiester bond it is very strong and complex while synthesis.

It forms an active intermediate complex, which able to form a bond by two sequential steps, in the first step the super call form of DNA get relaxed and by using of energy ligase catalyzes and form phosphodiester bond between 3'OH of deoxyribose sugar in nucleotide and 5'P of adjacent nucleotide.

Mechanism of DNA ligase:-

1. Two covalent phosphodiester bonds are formed between adjacent nucleotides, 3:OH is acceptor and 5'P is donor.

2. AMP is added to the enzyme active site lysine residue, pyrophosphate is reelased.

3. Transfer of AMP to the 5' region and form pyrophosphate bond.

4. Adjacent fragments are sealed.

Types of DNA ligases: there are three types of DNA ligases , Ecoli, T4DNa, mammalian..among them T4 DNA ligase is extensively used in molecular research.

​​​​​​ it plays an important role in the development of rDNA, by joining of desired gene fragment with vector DNA. Can work with both sticky ends and blunt end Ligation.