DNA Ligase and DNA Repair

Sloan-Kettering - Molecular Biology: Stewart Shuman: "ATP-dependent DNA ligases catalyze the joining of a 5'-phosphate-terminated strand to a 3'-hydroxyl-terminated strand via 3 sequential nucleotidyl transfer reactions. In the first step, ligase attacks the alpha-phosphorus of ATP to release PPi and form a covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide (P-N) bond to a lysine side chain on the enzyme. In the second step, the AMP is transferred to the 5'-end of the 5'-phosphate-terminated DNA strand to form a DNA-adenylate intermediate, A(5')pp(5')N. In the third step, ligase catalyzes attack by the 3'-OH of the nick on DNA-adenylate to join the 2 polynucleotides and release AMP.


ATP-dependent ligases are ubiquitous in eukaryotes; they are also encoded by certain eubacteria, bacteriophages, and eukaryotic DNA viruses. Sequence comparisons suggest that a catalytic domain common to all ATP-dependent ligases is embellished by additional isozyme-specific protein segments at the amino or carboxyl termini. The catalytic domain includes a set of 6 collinear motifs (I, III, IIIa, IV, V, and VI) that define a superfamily of covalent nucleotidyl transferases, encompassing the ATP-dependent polynucleotide ligases and GTP-dependent mRNA capping enzymes.


The nucleotide binding pocket of DNA ligase is composed of motifs I, III, IIIa, IV, and V. The lysine in motif I (KxDGxR) is the site of covalent attachment of AMP to the enzyme. Crystallography and mutagenesis have illuminated several of the enzymic functional groups that are involved in forming the ligase-adenylate intermediate."