CHLORELLA VIRUS-DNA LIGASE - NICK RECOGNITION AND MUTATIONAL ANALYSIS

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisti ng of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated st rand annealed to a bridging DNA template strand, The enzyme discrimina tes at the DNA binding step between substrates containing a 5'-phospha te versus a 5'-hydroxyl at the nick, Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for th e conserved Lys, Asp and Arg moieties at different steps of the ligase reaction, Mutant K27A is unable to form the covalent ligase-(Lys-epsi lon N-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate, Nonetheles s, K27A catalyzes phosphodiester bond formation at a pre-adenylated ni ck, This shows that the active site lysine is not required for the str and closure reaction, K27A binds to nicked DNA-adenylate, but not to a standard DNA nick, This suggests that occupancy of the AMP binding po cket of DNA ligase is important for nick recognition, Mutant D29A is a ctive in enzyme-adenylate formation and binds readily to nicked DNA, b ut 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 n icked DNA.