Role of nucleotidyl transferase motif V in strand joining by Chlorella virus DNA ligase Journal Article
| Authors: | Sriskanda, V.; Shuman, S. |
| Article Title: | Role of nucleotidyl transferase motif V in strand joining by Chlorella virus DNA ligase |
| Abstract: | ATP-dependent DNA ligases, NAD+-dependent DNA ligases, and GTP-dependent RNA capping enzymes are members of a covalent nucleotidyl transferase super-family defined by a common fold and a set of conserved peptide motifs. Here we examined the role of nucleotidyl transferase motif V (184LLKMKQFKDAEAT196) in the nick joining reaction of Chlorella virus DNA ligase, an exemplary ATP-dependent enzyme. We found that alanine substitutions at Lys186, Lys188, Asp192, and Glu194 reduced ligase specific activity by at least an order of magnitude, whereas substitutions at Lys191 and Thr196 were benign. The K186A, D192A, and E194A changes had no effect on the rate of single-turnover nick joining by preformed ligase-adenylate but affected subsequent rounds of nick joining at the ligase adenylation step. Conservative substitutions K186R, D192E, and E194D partially restored activity, whereas K186Q, D192N, and E194Q substitutions did not. Alanine mutation of Lys188 elicited distinctive catalytic defects, whereby single-turnover nick joining by K188A-adenylate was slowed by an order of magnitude, and high levels of the DNA-adenylate intermediate accumulated. The rate of phosphodiester bond formation at a pre-adenylated nick (step 3 of the ligation pathway) was slowed by the K188A change. Replacement of Lys188 by arginine reversed the step 3 arrest, whereas glutamine substitution was ineffective. Gel-shift analysis showed that the Lys188 mutants bound stably to DNA-adenylate. We infer that Lys188 is involved in the chemical step of phosphodiester bond formation. |
| Keywords: | gene mutation; mutation; mutation, missense; nonhuman; protein motif; amino acid substitution; protein binding; dose-response relationship, drug; time factors; dna; amino acid sequence; molecular sequence data; kinetics; protein structure, tertiary; threonine; dna mutational analysis; alanine; amino acids; adenosine triphosphate; molecular biology; point mutation; polydeoxyribonucleotide synthase; biochemistry; guanosine triphosphate; aspartic acid; mutagenesis; genetic conservation; diseases; glutamine; lysine; amino acid motifs; electrophoresis, polyacrylamide gel; chemical bond; multigene family; chlorella virus; dna ligases; viral proteins; adenylation; nucleotidyltransferase; dna nucleotidyltransferases; viruses; chemical bonds; bond formation; substitution reactions; adenosinetriphosphate; priority journal; article; phosphorus derivative |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 277 |
| Issue: | 12 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2002-03-22 |
| Start Page: | 9661 |
| End Page: | 9667 |
| Language: | English |
| DOI: | 10.1074/jbc.M110613200 |
| PUBMED: | 11751916 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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