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-straightepsilonN-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.
Sriskanda
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Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Shuman
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D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010710:Phosphates; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins
D010446:Peptide Fragments; D010710:Phosphates; D014764:Viral Proteins; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004247:DNA; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases