NAD+-dependent DNA ligase encoded by a eukaryotic virus.
We report the production, purification, and characterization 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'-PO(4) 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 (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) 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(+).
Sriskanda
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Moyer
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RW
Shuman
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D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D011088:DNA Ligases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D017353:Gene Deletion; D005822:Genetic Vectors; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D010802:Phylogeny; D011212:Poxviridae / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers
D009243:NAD; D001224:Aspartic Acid; D014443:Tyrosine; D011088:DNA Ligases; D015032:Zinc; D003545:Cysteine; D000409:Alanine