This research is designed to employ the eucaryotic virus vaccinia for the examination of the mechanism of action of several enzymes and structural proteins with DNA. Three enzymes with a putative role in DNA metabolism will be studied in detail, all of them being virus-specific. These include a type I topoisomerase (topo I), a type II topoisomerase (topo II), and an enzyme which catalyzes the nicking and crosslinking of the strands of the viral DNA (nicking-joining enzyme). These enzymes are all DNA binding proteins, and all are present in purified virus particles. Topo I has previously been purified to homogeneity. Covalent intermediates in the reaction with DNA will be identified and isolated and the sequence specificity of the binding site determined. Monoclonal antibodies will be obtained against the enzyme and these will be used to assess its role in transcription and in DNA replication. Topo II and the nicking-joining enzyme will be purified to homogeneity, using affinity chromatography and gel filtration. The physical properties of the purified enzymes will be documented, covalent intermediates with DNA will be isolated, and site specificity of binding and incision will be ascertained. The environmental variables significant for catalysis will be determined, including optima in ionic strength, temperature, and pH; specific ions; and cofactors. Monoclonal antibodies will be obtained against these enzymes and, as with the type I topoisomerase, used to investigate their roles in transcription and DNA replication. The viral type I topo gene will be mapped, using in vitro translation of hybrid-selected mRNA and detection by both enzyme activity and immunoprecipitation. In addition to these three enzymes, previous investigations of two viral DNA-binding structural proteins will be continued. Antisera have been obtained against both of these, an 11K dalton protein and a 24K dalton protein. The 11K protein will be mapped in the vaccinia genome, with the eventual objective of mapping the late promoter sequences. The physical and DNA-binding properties of the 24K polypeptide will be determined. The binding to superhelical DNA of increasing supercoiling will be used to assess the relative strength of binding to the native and denatured forms of DNA. These experiments will help lay a foundation for understanding the morphogenesis of this virus, the host cell lines of which are of mammalian origins.
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