DNA Topoisomerases and DNA Topology in E. coli

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0090880ZechiedrichTopoisomerases are ubiquitous, essential enzymes that pass DNA strands through each other to affect nearly every aspect of nucleic acid metabolism. Topoisomerase IV has been shown by the P.I. and others to be required for segregation of daughter chromosomes and modulation of DNA supercoiling in E. coli. The objectives of this project are to continue determining, using assays developed by the P.I., the roles and molecular mechanisms of DNA topoisomerases and to assess the consequences of unresolved topological structures in E. coli. Biochemical and genetic approaches with unknotting assays using site-specific recombination and topoisomerase inhibition allow us to test the hypotheses that topoisomerase IV unknots DNA and that persistent knots in the cell are lethal. Specifically, the objectives of this project are: (1) To ascertain which topoisomerase unknots DNA in vivo; (2) To determine how topoisomerase IV distinguishes different topological forms; and (3) To assess the consequences of unresolved DNA knots. E. coli is studied because it is well characterized with regard to topoisomerases, and biochemical and genetic analyses are straightforward with the genome sequence known. Results from the research will reveal how topoisomerases untangle rather than tangle DNA and how they distinguish problematic DNA knots or catenanes from beneficial DNA supercoils.