The current focus of our research program is on the structure, function, and mechanism of DNA topoisomerases. These are essential enzymes which can modulate the topological structure of DNA and play important roles in all aspects of chromosome functions including replication, transcription, recombination, repair and segregation. DNA topoisomerases can allow DNA strands to pass freely through each other, the mechanism by which this is achieved is an important question at the interface of chemistry and biology. Furthermore, DNA topoisomerases are important pharmacological targets for many clinically useful antibiotics and anti-cancer drugs. Studies on the mechanism of topoisomerase can provide new insights into cancer chemotherapy. These topoisomerase targeting drugs are useful reagents in probing the mechanism and function of these enzymes. The proposed mechanistic analysis of topoisomerase is centered on two different types of enzymes: topoisomerase II and III. Topoisomerase II requires ATP for its stand passage activity. The proposed experiments will address the mechanism of ATP binding and hydrolysis in the conformation transition resulting in protein clamp closure. Site-specific mutants for the critical residues that can affect this process will be generated and analyzed in detail. Topoisomerase III is recently cloned from Drosophila cells and will be over-produced and purified. We will develop a reaction in annealing the single strand RNA circles with complementary sequences to assay for the possible RNA topoisomerase activity associated with topoisomerase III. The function of topoisomerase I will be approached from two directions. One is to use mutants and transgenic fly strains generated in our lab to probe its function in oogenesis, early embryonic development, and late larval development. The other is to identify the proteins that can interact with the N-terminal, hydrophilic domains of topoisomerase I, a region which we have shown to be able to target a protein to the transcriptionally active loci. Both biochemical and genetic techniques will be applied to identify the protein components involved in the targeting of topoisomerase I to a chromatin region engaged in transcription.
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