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. As DNA is replicated and transcribed, the twisting, turning, tangling, and untangling of the duplex strands of DNA 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. hematopoiesis) and bacteria (i.e. in adapting to a harsh environment). A method that uses the gamma delta 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 the resolution system, supercoiling domains have been shown to be abundant and place stochastically over 10% of the bacterial chromosome. For cells growing exponentially, the probability that two sites in a chromosome will interact through supercoil movement fits a first order function; there is a 50% probability that a barrier will exist for each 15 kb of distance separating two sites. In this proposal there are three specific aims. First, the global pattern of domain structure will be studied by expanding the survey to cover 30% of the genome. Two critical points of cell division control will be included in the survey the origin and terminus of DNA replication. Second, using a kinetic analysis of the strand exchange process, these studies will count barriers that stop dynamic DNA movement along the chromosome. In addition, a genetic screen will be carried out to find the set of genes that modulate the number of domains. Two essential genes in bacteria, DNA gyrase and Topoisomerase IV, have both turned up in this screen. Other genes will be mapped and characterized.
The third aim i nvolves the theory that knots and tangling of DNA strands pose a major impediment to long range DNA dynamics. Special transposons will be built to find and count knots in different regions of the chromosome.
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