DNA ligases catalyze phosphodiester bond formation via an enzyme-AMP intermediate requiring adenylation. The source of AMP differentiates ligase families. Bacteriophage T4 ligase and most eukaryotic ligases are ATP-dependent, cleaving ATP to AMP and pyrophosphate to activate the active-site lysine. In contrast, Escherichia coli DNA ligase and several other bacterial ligases are NAD+-dependent, using oxidized nicotinamide adenine dinucleotide as adenyl donor and releasing NMN. Both classes require divalent magnesium for catalysis. Recognizing cofactor specificity is essential for choosing buffe
Ref:
NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.