MECHANISMS AND TRANSITION STATES FOR DNA GLYCOSYLASES

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The long term goal of this research is to obtain a fundamental understanding how DNA repair glycosylases catalyze the hydrolysis of damaged pyrimidine and purine bases from DNA. The immediate goal of this proposal is to understand the mechanism and catalysis of the pyrimidine specific enzyme, uracil DNA glycosylase (UDG) from E. coli. UDG is of health related interest because of its role in removing premutagenic uracil bases from DNA, and is also a viable target for anti-viral drugs, because of its essential role in viral latency/reactivation cycles and viral DNA replication. The specific aims of this research are to i) elucidate the complete kinetic and thermodynamic mechanism of the UDG reaction ii) determine the structures for enzyme-bound substrate and products, iii) establish the chemical basis for the 1012-fold catalytic power of UDG, and iv) determine the transition-state structure for enzymatic and nonenzymatic hydrolysis of uracil from DNA. The microscopic steps along the reaction pathway will be characterized using rapid kinetic methods, including the suggested key step in uracil recognition-the "flipping out" of the base from the DNA helix. A novel fluorescence assay has been developed for much of this kinetic work. The structures of the enzyme-bound DNA substrate and products will be solved by X-ray crystallography, as we have already done for the free enzyme. The chemical mechanism of the enzymatic reaction will be examined by mutagenesis of conserved active-site residues seen in our partial crystal structure of the free enzyme. The damaging effects of these mutations on the microscopic kinetic steps, binding parameters, and the pH-rate profiles will be quantified. Thus, the catalytic roles of these residues and their pKa values will be suggested. Heteronuclear NMR spectroscopy will then be used directly to determine the side-chain pKa values of key groups identified by mutagenesis, establishing a thermodynamic framework for the proton transfers involved in the reaction. The transition-state structure will be characterized on the basis of dual-label competitive isotope effect measurements. For the isotope effect work, a new and efficient method for the enzymatic synthesis of isotopically labeled deoxyuridine- containing DNA will be used. The enzymatic isotope effects and transition-state will them be compared with the water and acid-catalyzed hydrolysis of uracil from isotopically labeled DNA. It is anticipated that this work will provide a basic understanding of the factors that contribute to stabilization of the enzymatic transition-state for hydrolysis of pyrimidine bases from DNA.