AN ETOPOSIDE-INDUCED BLOCK IN VACCINIA VIRUS TELOMERE RESOLUTION IS DEPENDENT ON THE VIRUS-ENCODED DNA-LIGASE

Etoposide, an inhibitor of the breakage-reunion reaction associated wi th cellular type II DNA topoisomerases, was shown to inhibit plaque fo rmation of vaccinia virus. This drug had a major effect on the segrega tion of newly replicated DNA concatemers. Gene expression and the init iation and elongation phases of viral DNA replication were essentially unaffected. Pulsed-field gel electrophoresis of viral DNA replicated in the presence of etoposide revealed two major classes of DNA: the ma ture monomeric linear genome and DNA that failed to enter the gel (the relative proportions depending on the concentrations of drug). Restri ction enzyme analysis showed a severe defect in telomere resolution. I n addition, slowly migrating restriction fragments were suggestive of a general recombination defect. We have isolated several etoposide-res istant mutants and used marker rescue and DNA sequencing to localize t he resistance-causing mutation to the amino terminus of the viral DNA ligase gene. Inactivation of the DNA ligase also resulted in an etopos ide-resistant phenotype, but to a lesser extent. The telomere resoluti on and segregation defects were corrected both in the drug-resistant m utants and in the DNA ligase knockout mutants. Reinsertion of wild-typ e or mutant DNA ligase in the viral thymidine kinase locus confirmed t he role of the viral DNA ligase in conferring sensitivity not only to etoposide but also to another topoisomerase II inhibitor, 4'-(9-acridi nylamino) methanesulphon-m-anisidide (mAMSA). The data suggest that th e nonessential DNA ligase is involved in telomere resolution, possibly as part of a general recombinase.