The long-term objective of the proposed research is to understand the molecular mechanisms involved in regulation of DNA conformation. Regulation of DNA conformation is likely to be an integral part of genetic control mechanisms and its understanding may have broad implications in many areas of health-related genetic research. In the past, DNA topoisomerases have been identified as the major enzymes involved in regulating DNA conformation; More recently, accumulating evidence has indicated that significant local DNA conformational changes can be brought about by DNA helix-tracking processes such as transcription elongation and the action of certain DNA helix-tracking proteins including SV40 T antigen, E. coli UvrAB and some partially characterized DNA helix-tracking proteins from Xenopus laevis and Saccharomyces cerevisiae. In the present proposal, we will focus our studies on the regulation of local DNA conformation and its potential role in several genetic processes. A major part of the proposed research is to test whether the variable rates of movement of RNA polymerases along the DNA template can generate a high degree of local supercoiling which influences template functions in initiation of DNA replication, recombination and repair. We will also test whether anchored translocation is another means to regulate local DNA conformation. The possibility that E. coli UvrAB may alter local DNA conformation by a mechanism involving anchored translocation will be investigated. Finally, using an assay specific for DNA helix-tracking proteins, we will purify the DNA helix-tracking protein(s) from Saccharomyces cerevisiae and investigate its mechanism of translocation on duplex DNA.
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