Mutational Analysis of E coli DNA Topoisomerase III

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DESCRIPTION (Provided by applicant): DNA topoisomerases are enzymes that alter the linking number of DNA and, as such, are responsible for topological inter- and intra-conversions within DNA molecules. These conversions include the knotting or unknotting of DNA, the supercoiling or relaxation of DNA (an intra-molecular conversion) and the catenation (or decatenation) of DNA (an inter-molecular conversion). Topoisomerases can be classified into two major categories, type I and type II. The type I enzymes can be further subdivided into type IA prokaryotic DNA topoisomerase I (Topo I) and III (Topo III), and eukaryotic DNA topoisomerase III (Topo III)) and type IB (eukaryotic DNA topoisomerase I and bacteriophage recombinases). It has been shown that Topo III-like activities appear to function in homologous pathways in both prokaryotes and eukaryotes. In particular, it is becoming increasingly evident that Topo III-like enzymes interact with DNA helicases and act in the suppression of recombination in both yeast and E. coli. Topo III of E. coli is extremely amenable to both biochemical and genetic characterization; therefore, the characterization of E. coli Topo III has been very useful to establishing the role of these enzymes in the DNA metabolism of both prokaryotes and eukaryotes. The specific aims of this grant are: 1. Further identify residues involved in E. coli Topo III-mediated catalysis and elucidate the mechanism of type IA topoisomerase-mediated catalysis 2. Elucidate the role of E. coli Topo III in recombination 3. Identify and examine the role of topoisomerase III interactions with other cellular proteins, in particular, DNA helicases.