19 DNA Ligases

DNA ligases are Mg++-dependent enzymes that catalyze the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA (for review and main references up to 1992, see Engler and Richardson 1982; Lindahl and Barnes 1992). The first step in the reaction is the formation of a covalent enzyme/adenylate intermediate. DNA ligases from eukaryotes, archaea, and viruses employ ATP as cofactor, whereas eubacterial DNA ligases use NAD to generate the adenylyl group. The ATP is cleaved to AMP and pyrophosphate with the adenylyl residue linked by a phosphoramidate bond to the &-amino group of a specific lysine residue at the active site of the protein. The reaction is readily reversed in vitro by addition of pyrophosphate. Since DNA ligases contain an unusually reactive lysine residue in their active site, a Schiff base can be formed with pyridoxal phosphate. In consequence, the activities of DNA ligases, e.g., mammalian DNA ligases I and 11, are inhibited in vitro by pyridoxal phosphate. ATP-dependent DNA ligases can employ certain cofactor analogs such as dATP, but the anomalous enzyme/nucleotide complexes formed by some ligases appear to function poorly in subsequent steps of the DNA-joining reaction. The activated AMP residue of the DNA ligase/adenylate intermediate is transferred to the 5 -phosphate terminus of a single-strand break in double-stranded DNA to generate a covalent DNA-AMP complex with a 5'-5' phosphoanhydride bond. In the final step of DNA ligation, unadenylylated DNA ligase is required for the generation of a phosphodiester bond and catalyzes displacement of the AMP residue through attack by the adjacent 3 -hydroxyl group on the adenylylated site (Fig. 1). The active site of mammalian DNA ligase I has been identified by isolation and amino acid sequencing of the tryptic peptide containing a ly~ine-[~H]AMP moiety and localized by comparison with the complete peptide sequence deduced from the cDNA. The amino acid sequence of the peptide containing the active-site lysine residue has also been