Vaccinia virus DNA ligase: specificity, fidelity, and inhibition.
Vaccinia DNA ligase and mammalian DNA ligases II and III comprise a distinct subgroup of structurally homologous enzymes within the eukaryotic DNA ligase family. The specificity and fidelity of the viral enzyme were investigated using purified recombinant ligase and synthetic duplex DNA substrates containing a single strand discontinuity. Vaccinia ligase catalyzed efficient strand joining on nicked DNAs in the presence of magnesium and ATP (Km = 95 microM). dATP, ITP, AMPPCP, 3'dATP, and ATP alpha S could not substitute for ATP; of these, 3'dATP and ATP alpha S were inhibitors of ligation. The vaccinia enzyme was unable to seal strands across a 1 nt (nucleotide) or 2 nt gap. Ligase action at a 1 nt gap resulted in accumulation of high levels of the normally undetectable DNA-adenylate reaction intermediate. In contrast, no DNA-adenylate was formed at a 2 nt gap. A native gel mobility shift assay showed that vaccinia DNA ligase was capable of discriminating between nicked and gapped DNAs at the substrate binding step. The ligase was fairly tolerant of mismatches at a nick involving the 5' phosphate donor terminus but was inhibited strongly by mismatches at the 3' OH acceptor terminus, especially by purine.-purine mispairs. These findings underscore the importance of a proper 3' OH terminus in substrate recognition and reaction chemistry but also raise the possibility that ligase may generate mutations during DNA repair by sealing DNA molecules with mispaired ends. Ligase was inhibited by several DNA binding drugs, including, in order of decreasing potency, distamycin, ethidium bromide, and actinomycin. Strand joining by purified ligase was not affected by etoposide, a drug that inhibits vaccinia virus replication in vivo and which depends on the presence of vaccinia ligase for its antiviral action.
Shuman
S
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004214:Distamycins / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D009675:Novobiocin / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000494:pharmacology; D014616:Vaccinia virus / Q000201:enzymology*
D004214:Distamycins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D009675:Novobiocin; D003609:Dactinomycin; C030000:stallimycin; D000255:Adenosine Triphosphate; D011088:DNA Ligases; D004996:Ethidium