Mechanistic Biology of Topoisomerase 1B

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DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how type IB DNA topoisomerases (topo) catalyze reversible DNA strand cleavage and religation, and how the chemical and dynamic nature of the phosphotyrosyI-DNA covalent complex promotes such important biological processes as DNA strand transfer, recombination, supercoil unwinding, and anticancer drug binding. In this work, we will employ the small, sequence specific type 1B topo from vaccinia virus because it is the only type IB enzyme amenable to detailed NMR, fluorescence, and kinetic and thermodynamic studies. The specific aims are as follows: (i) Determine the basis for specific recognition of CCCTT sites in DNA. The importance of base and phosphodiester interactions in specificity will be evaluated using novel base analogs and nonbridging methylphosphonate substitutions, respectively. (ii) Elucidate the mechanism of nucleophilic catalysis. The catalytic interactions of the scissile phosphodiester will be dissected using the combined approach of mutagenesis, nonbridging phosphorothioate substitutions. Novel solid-state REDOR NMR structural methods will be used to confirm these interactions. (iii) Understand how Topo I removes supercoils from DNA. Topo I must release its grip on DNA to allow supercoil relaxation to occur. To elucidate these critical motions, 19F-labeled DNA molecules and NMR spectroscopy will be used to measure the dynamics of the DNA within the covalent complex. In addition, we will use a small supercoiled plasmid with a single cleavage site, to evaluate the role of three key variables on the supercoil relaxation mechanism: the DNA superhelical density, the lifetime of the covalent complex, and the "tightness" of the enzyme grip on the rotating DNA. We anticipate these measurements will guide our efforts to design inhibitors of Topo I and to engineer the enzyme to perform other useful DNA transformations.