NAD+-dependent DNA ligase encoded by a eukaryotic virus Journal Article
| Authors: | Sriskanda, V.; Moyer, R. W.; Shuman, S. |
| Article Title: | NAD+-dependent DNA ligase encoded by a eukaryotic virus |
| Abstract: | We report the production, purification, and character. ization of an NAD+-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD+ ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD+ ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD+. Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD+. Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD+ ligases. Deletion of domain Ia of AMEPV ligase abolished the sealing of 3′-OH/5′ -PO4 nicks and the reaction with NAD+ to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr39, Tyr40, Asp48, and Asp 52) or abolished (Tyr51) sealing of a 5′-PO 4 nick and adenylyl transfer from NAD+ without affecting ligation of DNA-adenylate. We conclude that: (i) NAD+-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD+. |
| Keywords: | controlled study; gene deletion; genetics; mutation; dose response; nonhuman; molecular genetics; protein domain; protein motif; metabolism; biological model; models, biological; amino acid substitution; protein degradation; protein binding; dose-response relationship, drug; tyrosine; bacteria (microorganisms); time; time factors; gene vector; genetic vectors; evolution, molecular; biosynthesis; chemistry; dna; molecular evolution; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; enzyme analysis; dna viruses; eukaryota; nucleotide sequence; substrate specificity; base sequence; amino acid; protein structure, tertiary; alanine; catalysis; adenosine triphosphate; sequence homology; zinc; structure analysis; polydeoxyribonucleotide synthase; enzyme specificity; biochemistry; isolation and purification; zinc finger protein; phosphate; protein tertiary structure; aspartic acid; nad; cysteine; eukaryote; amino acid motifs; nicotinamide adenine dinucleotide; polyacrylamide gel electrophoresis; electrophoresis, polyacrylamide gel; dna ligases; phylogeny; nucleotidyltransferase; poxvirus; poxviridae; zinc fingers; bacteria; nucleoside triphosphate; phylogenetic tree; eubacterium; deoxyadenosine triphosphate; monomers; positive ions; entomopoxvirus; substitution reactions; divalent cations; priority journal; article; unidentified entomopoxvirus; amsacta moorei; amsacta moorei entomopoxvirus; amsacta |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 39 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-09-28 |
| Start Page: | 36100 |
| End Page: | 36109 |
| Language: | English |
| DOI: | 10.1074/jbc.M105643200 |
| PUBMED: | 11459847 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
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