Chlorella virus DNA ligase: Nick recognition and mutational analysis Journal Article
| Authors: | Sriskanda, V.; Shuman, S. |
| Article Title: | Chlorella virus DNA ligase: Nick recognition and mutational analysis |
| Abstract: | 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-εN-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 preadenylated 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. |
| Keywords: | mutation; nonhuman; enzyme activity; structure-activity relationship; dna; amino acid sequence; conserved sequence; kinetics; nucleotide sequence; peptide fragments; substrate specificity; mutagenesis, site-directed; binding sites; adenosine triphosphate; dna binding; polydeoxyribonucleotide synthase; phosphates; dna nick translation; chlorella virus; paramecium bursaria chlorella virus 1; dna ligases; viral proteins; adenylation; virus dna; dna template; adenosine monophosphate; chlorella; phocidae; priority journal; article |
| Journal Title: | Nucleic Acids Research |
| Volume: | 26 |
| Issue: | 2 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 1998-01-01 |
| Start Page: | 525 |
| End Page: | 531 |
| Language: | English |
| DOI: | 10.1093/nar/26.2.502 |
| PUBMED: | 9421510 |
| PROVIDER: | scopus |
| PMCID: | PMC147278 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 12 December 2016 -- Source: Scopus |
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