Related Experiment Videos
An etoposide-induced block in vaccinia virus telomere resolution is dependent on the virus-encoded DNA ligase
A M DeLange1, M S Carpenter, J Choy
1Department of Human Genetics, University of Manitoba, Winnipeg, Canada.
Abstract:
Etoposide, an inhibitor of the breakage-reunion reaction associated with cellular type II DNA topoisomerases, was shown to inhibit plaque formation of vaccinia virus. This drug had a major effect on the segregation of newly replicated DNA concatemers. Gene expression and the initiation 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 mature monomeric linear genome and DNA that failed to enter the gel (the relative proportions depending on the concentrations of drug). Restriction enzyme analysis showed a severe defect in telomere resolution. In addition, slowly migrating restriction fragments were suggestive of a general recombination defect. We have isolated several etoposide-resistant mutants and used marker rescue and DNA sequencing to localize the resistance-causing mutation to the amino terminus of the viral DNA ligase gene. Inactivation of the DNA ligase also resulted in an etoposide-resistant phenotype, but to a lesser extent. The telomere resolution and segregation defects were corrected both in the drug-resistant mutants and in the DNA ligase knockout mutants. Reinsertion of wild-type or mutant DNA ligase in the viral thymidine kinase locus confirmed the role of the viral DNA ligase in conferring sensitivity not only to etoposide but also to another topoisomerase II inhibitor, 4'-(9-acridinylamino) methanesulphon-m-anisidide (mAMSA). The data suggest that the nonessential DNA ligase is involved in telomere resolution, possibly as part of a general recombinase.
Insights
Etoposide inhibits vaccinia virus plaque formation by affecting DNA segregation and telomere resolution. Mutations in viral DNA ligase confer resistance, suggesting its role in resolving DNA ends during replication.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Etoposide targets type II DNA topoisomerases, crucial for DNA replication and segregation.
- Vaccinia virus replication involves complex DNA processing, including telomere resolution.
Purpose of the Study:
- Investigate the mechanism of etoposide's inhibition of vaccinia virus plaque formation.
- Identify viral factors involved in etoposide sensitivity and resistance.
Main Methods:
- Plaque assays, pulsed-field gel electrophoresis, and restriction enzyme analysis were used to study viral DNA.
- Isolation and characterization of etoposide-resistant mutants, including marker rescue and DNA sequencing.
- Gene knockout and reinsertion experiments to confirm the role of viral DNA ligase.
Main Results:
- Etoposide inhibited vaccinia virus plaque formation and disrupted DNA segregation and telomere resolution.
- Etoposide resistance mapped to mutations in the viral DNA ligase gene.
- Viral DNA ligase inactivation conferred etoposide resistance and corrected segregation/telomere resolution defects.
Conclusions:
- Viral DNA ligase plays a critical role in telomere resolution and DNA segregation during vaccinia virus replication.
- The DNA ligase is involved in sensitivity to etoposide and other topoisomerase II inhibitors.
- Viral DNA ligase may function as a general recombinase in resolving DNA ends.