Chlorella virus DNA ligase:: nick recognition and mutational analysis

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand, The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick, Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction, Mutant K27A is unable to form the covalent ligase-(Lys-epsilon N-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate, Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick, This shows that the active site lysine is not required for the strand closure reaction, K27A binds to nicked DNA-adenylate, but not to a standard DNA nick, This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition, Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation, R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.