Characterization of mimivirus NAD+-dependent DNA ligase.
Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.
Benarroch
Delphine
D
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Shuman
Stewart
S
D000048:Acanthamoeba / Q000821:virology*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017996:Entomopoxvirinae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D011088:DNA Ligases; C120640:DNA ligase (NAD); D003545:Cysteine