Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme Journal Article
| Authors: | Sekiguchi, J.; Shuman, S. |
| Article Title: | Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme |
| Abstract: | Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active- site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes. |
| Keywords: | mutation; binding affinity; dna repair; enzyme activity; vaccinia virus; binding site; binding sites; catalysis; polydeoxyribonucleotide synthase; guanosine triphosphate; phosphates; dna determination; dna nick translation; polyacrylamide gel electrophoresis; dna topoisomerases, type i; dna ligases; adenosine phosphate; dna nucleotidyltransferases; adenosine monophosphate; priority journal; article |
| Journal Title: | Journal of Virology |
| Volume: | 71 |
| Issue: | 12 |
| ISSN: | 0022-538X |
| Publisher: | American Society for Microbiology |
| Date Published: | 1997-12-01 |
| Start Page: | 9679 |
| End Page: | 9684 |
| Language: | English |
| PUBMED: | 9371633 |
| PROVIDER: | scopus |
| PMCID: | PMC230277 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 17 March 2017 -- Source: Scopus |
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