Effects of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase.

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RESUMO

Vaccinia virus cores contain a type I topoisomerase which promotes the relaxation of superhelical DNA of either handedness (Bauer et al., Proc. Natl. Acad. Sci. U.S.A. 74:1841-1845, 1977). The activity of partially purified vaccinia virus topoisomerase (VV-Topo I) was determined in the presence of ATP, dATP, GTP, ADP, and ATP analogs in which hydrolysis of the alpha, beta or beta, gamma phosphate bond is restricted. Topoisomerase activity was stimulated 2.5-fold by the addition of 2 to 4 mM ATP or dATP to standard assay mixtures; 2 mM GTP produced no significant effect on enzyme activity. The addition of 2 mM beta, gamma-imido ATP or 2 mM gamma-thiophosphate ATP reduced VV-Topo I activity by 80 and 65%, respectively. In contrast, 4 mM alpha, beta-methylene ATP produced no significant change in topoisomerase activity compared to ATP itself. Assays performed in the presence of 4 mM ADP exhibited an 80% reduction in enzyme activity. The preparations of VV-Topo I used for these studies showed, however, no detectable DNA-dependent or -independent ATPase activity. The activity of VV-Topo I was similarly measured in the presence of the antibiotics novobiocin and coumermycin A1, which inhibited enzyme activity by 50% at concentrations of 180 and 40 microM, respectively. Comparable inhibition of VV-Topo I activity was observed in the presence of 1 mM beta, gamma-imido ATP. We determined that novobiocin inhibits vaccinia core transcription at the same concentrations which inhibit vaccinia core topoisomerase I activity. These results suggest that the vaccinia DNA topoisomerase may play a role in the ATP-dependent transcription of viral genes from intact core particles.

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