DNA domain formation is critical for eukaryotic and prokaryotic cells alike. The dynamics of DNA movement inside a living cell is a central problem in biology. How DNA is twisted, turned, tangled, and untangled is a major problem that impinges on cellular enzymes that perform functions like transcription, genetic recombination, chromosome segregation, and replication. Domain regulation underpins cell development and gene regulation in organisms as diverse as man (i.e. hematopoesis) and bacteria (i.e. in adapting to a harsh environment). A method that uses the Tn_ site-specific recombination pathway has been developed to study supercoil dynamics and domain structure inside living cells. This analysis can be performed at any desired point in the bacterial genome.Using our system, several types of domain boundaries have been located. The most abundant barrier class occurs during replication and these barriers are located stochastically over the sequence with an average spacing of 30 kb. Rarer sequence specific barriers arise from transcription at very active promoter and at gene clusters that encode membrane-inserted proteins. We plan to derive the global pattern of domain structure by analyzing at least 50 test intervals that span the genomes of E. coli and S. enterica serovar Typhimurium.
One aim i s to see if conserved operons that encode clusters of membrane proteins are position specific barriers. There are 23 of these operons in the sequenced genomes of E. coIi and Salmonella that have remained at fixed points in the drifting genomes. Second, we will identify mutants that change DNA domain structure at two critical points for controlling cell division-- the origin and terminus of DNA replication. Third, we will characterize proteins that alter DNA dynamics. The results from these studies will provide critical information about how DNA dynamics influences a wide variety of biochemical processes on DNA. They will also shed light on the mechanisms by which chromosomes achieve structural stability over deep time.
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