The goal of this proposed research is to understand the roles and mechanisms of DNA topoisomerases in regulating DNA conformations and various genetic processes. Three areas of research will be focused. (1) DNA conformation during RNA transcription. Many recent findings have led us to propose that the transcriptional process can generate positive supercoils ahead of and negative supercoils behind the transcribing RNA polymerase. The degree of steady state supercoiling induced by transcription depends not only on the presence of multiple DNA topoisomerases but also on the distribution, size and activity of the transcriptional units. This model will be tested in E. coli, yeasts and mammalian cells by a variety of genetic and biochemcial methods. (2) DNA topology and the roles of DNA topoisomerases in DNA replication. The multiple and differential roles of mammalian DNA topoisomerase I and II in different stages of SV40 chromatin replication will be studied in vitro. We propose a twin- supercoiling loop model for the helicase action of SV40 T antigen during initiation and elongation of SV40 DNA replication. This model will also be tested in vitro. (3) Regulation of DNA topoisomerases during cell growth and differentiation. We will study the transcriptional regulation of DNA topoisomerase genes in mouse BALB/c 3T3 cells during growth stimulation and in murine erythroleukemic cells during differentiation. The identification of cis- and trans-acting growth-regulated elements in topoisomerase gene systems may form a foundation for further study of the regulatory mechanisms of cell growth and differentiation.
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