NAD+-dependent DNA Ligase Encoded by a Eukaryotic Virus

• Published in: The Journal of biological chemistry 2001-09, Vol.276 (39), p.36100-36109
• Main Authors: Sriskanda, Verl, Moyer, Richard W., Shuman, Stewart
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Amino Acid Motifs; Amino Acid Sequence; Aspartic Acid - chemistry; Base Sequence; Catalysis; Cysteine - chemistry; DNA Ligases - biosynthesis; DNA Ligases - genetics; DNA Ligases - isolation & purification; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Evolution, Molecular; Gene Deletion; Genetic Vectors; Models, Biological; Molecular Sequence Data; Mutation; NAD - metabolism; Phylogeny; Poxviridae - genetics; Protein Binding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Substrate Specificity; Time Factors; Tyrosine - chemistry; Zinc - metabolism; Zinc Fingers
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M105643200

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′-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 Asp52) or abolished (Tyr51) sealing of a 5′-PO4 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+.