VACCINIA VIRUS-DNA LIGASE - SPECIFICITY, FIDELITY, AND INHIBITION

Vaccinia DNA ligase and mammalian DNA ligases II and III comprise a di stinct subgroup of structurally homologous enzymes within the eukaryot ic DNA ligase family. The specificity and fidelity of the viral enzyme were investigated using purified recombinant ligase and synthetic dup lex DNA substrates containing a single strand discontinuity. Vaccinia ligase catalyzed efficient strand joining on nicked DNAs in the presen ce of magnesium and ATP (K-m = 95 mu M). dATP, ITP, AMPPCP, 3'dATP, an d ATP alpha S could not substitute for ATP; of these, 3'dATP and ATP a lpha 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 und etectable DNA-adenylate reaction intermediate. In contrast, no DNA-ade nylate was formed at a 2 nt gap. A native gel mobility shift assay sho wed that vaccinia DNA ligase was capable of discriminating between nic ked and gapped DNAs at the substrate binding step. The ligase was fair ly tolerant of mismatches at a nick involving the 5' phosphate donor t erminus but was inhibited strongly by mismatches at the 3' OH acceptor terminus, especially by purine purine mispairs. These findings unders core the importance of a proper 3' OH terminus in substrate recognitio n and reaction chemistry but also raise the possibility that ligase ma y generate mutations during DNA repair by sealing DNA molecules with m ispaired ends. Ligase was inhibited by several DNA binding drugs, incl uding, in order of decreasing potency, distamycin, ethidium bromide, a nd 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 ac tion.