The research described here will investigate the nature of DNA supercoiling in living cells by exploring two parallel models: (A) Supercoiling as a dynamic property which can vary locally due to DNA tracking processes such as transcription; and (B) Supercoiling as a stable property of chromosomes which contributes to structural organization and compaction of the genome. Currently, the quantitative relationship between dynamic supercoiling and stable supercoiling in vivo is an unresolved question. Transcription can give rise to changes in the overall superhelix density of the chromosome, but the efficiency with which RNA polymerases introduce superturns is not yet understood. The ability of transcription to alter superhelical tension depends on many mechanistic factors such as the degree of anchoring of RNA polymerase, the rate at which supercoils diffuse along DNA, the kinetics of topoisomerase turnover, and the possible existence of topological barriers along the chromosome. Part of the research proposed here is based on a model system in which transcription can unwind the template to a negative superhelix density greater than or equal to 0.35 in tens of seconds. This rate approaches the maximal efficiency for an elongating RNA polymerase wherein transcription of ten basepairs introduces one positive superturn in front of and one negative superturn behind the transcription complex. In this model system, expression of a marker gene is strongly affected by the orientation of another adjacent, strongly-expressed gene. Experimentally, this system provides an estimate of the kinetics of topoisomerase enzymes in vivo. Further studies will elucidate some of the fundamental interactions which anchor RNA polymerase in cells. Another important element of this proposal is to understand the nature of stable DNA supercoiling in eukaryotes by directly probing for torsionally strained DNA on the chromosomes of yeast. This part of the proposal should provide insight into the existence of topological domains in eukaryotic chromosomes which will have important implications for the maintainenance of DNA supercoiling and gene expression in higher organisms. Finally, the stable topological structure of archaebacterial chromosomes and the question of whether DNA is positively supercoiled in vivo in the hyperthermophilic members of this group is a third major aspect of this research.
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