Title: “Control of DNA Topology”PI: Yuk-Ching Tse-Dinh, PhDProject Summary/AbstractThe long term goals of this project are to understand how the activity, regulation and interactions of DNAtopoisomerases control DNA topology and affect vital cellular functions. Drugs that target type IB and type IIAtopoisomerases are used in current anti-cancer and anti-bacterial therapy. Bacterial type IA topoisomeraseshave been validated as a useful target for discovery and development of novel antibacterial therapy to treatdrug resistant bacterial pathogens that cannot be eliminated with current antibiotics, including the antibioticsthat target bacterial topoisomerase II. The proposed research activities for the next funding period wouldelucidate the complete catalytic mechanism and provide new insights into the protein-protein interactions andregulation of Escherichia coli topoisomerase I activity. This information is needed to fully realize the potentialof discovery of novel antibacterial drugs specific for type IA topoisomerases that is present in every bacterialpathogen as a potential therapeutic target. Topoisomerase I catalyzes the relaxation of negatively supercoiledDNA by cleaving a single DNA strand in the underwound duplex DNA and passing the complementary DNAsingle strand through the break before religation of the cleaved strand to increase the DNA winding. Themolecular mechanism of the large enzyme conformational changes that are required for the coordinatedmovement of the passing DNA is the critical barrier for elucidating how bacterial topoisomerase I can relaxnegatively supercoiled DNA with high efficiency to prevent hypernegative DNA supercoiling and R-loopstabilization that can arise during transcription elongation. Structural and biochemical studies will beconducted to test hypotheses generated from crystal structures obtained in this project on the mechanism ofenzyme DNA conformational change and DNA passage. Interaction sites of endogenous bacterial toxins thatcan act as inhibitors of topoisomerase I catalytic activity will be identified. Our preliminary results showed thatdeacetylation of topoisomerase I may be an important function of the E. coli deacetylase CobB. Novelmechanisms of regulation of DNA topology and bacteria growth via acetylation-deacetylation of topoisomeraseI will be investigated. The project will include direct participations of co-Investigators who are experts instructural biology and single molecule studies of enzyme-DNA interactions. The results will provide themolecular basis of bacterial topoisomerase I function, and new insights into growth regulation of bacteria viamodulation of topoisomerase I enzyme activity. Success in these experiments would support the drugdiscovery efforts of utilizing bacterial topoisomerase I as a new target for antibiotics.
