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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Virology Study Section (VR)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
State University New York Stony Brook
Schools of Medicine
Stony Brook
United States
Zip Code
Benham, Craig J; Savitt, Anne G; Bauer, William R (2002) Extrusion of an imperfect palindrome to a cruciform in superhelical DNA: complete determination of energetics using a statistical mechanical model. J Mol Biol 316:563-81
Reddy, M K; Bauer, W R (1989) Activation of the vaccinia virus nicking-joining enzyme by trypsinization. J Biol Chem 264:443-9
Foglesong, P D (1989) Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays. Anal Biochem 182:284-8
Yang, W P; Kao, S Y; Bauer, W R (1988) Biosynthesis and post-translational cleavage of vaccinia virus structural protein VP8. Virology 167:585-90
Yang, W P; Bauer, W R (1988) Purification and characterization of vaccinia virus structural protein VP8. Virology 167:578-84
White, J H; Bauer, W R (1988) Applications of the twist difference to DNA structural analysis. Proc Natl Acad Sci U S A 85:772-6
Kao, S Y; Bauer, W R (1987) Biosynthesis and phosphorylation of vaccinia virus structural protein VP11. Virology 159:399-407
White, J H; Bauer, W R (1987) Superhelical DNA with local substructures. A generalization of the topological constraint in terms of the intersection number and the ladder-like correspondence surface. J Mol Biol 195:205-13
White, J H; Bauer, W R (1986) Calculation of the twist and the writhe for representative models of DNA. J Mol Biol 189:329-41
Armstrong, K A; Ohtsubo, H; Bauer, W R et al. (1986) Characterization of the gene products produced in minicells by pSM1, a derivative of R100. Mol Gen Genet 205:56-65

Showing the most recent 10 out of 14 publications