This proposal presents a research plan for a comparative mechanistic analysis of uracil DNA glycosylases (UDG's) from E. coli and Vaccina virus, and mismatch-specific uracil DNA glycosylase (MUG) from E. coli. Uracil is typically found in RNA and is not a normal DNA base. It may arise in DNA via the deamination of cytidine, or during replication by misincorporation of dUMP instead of TMP. The repair enzymes UDG and MUG function by catalyzing the hydrolysis of the C-N glycosidic bond between uracil and a 2'-deoxyridbose residue. X-ray structural analysis of the UDG's and MUG have shown that these enzymes mediate base-flipping, a novel protein-DNA interaction characterized by a bound state in which the dUMP residue is rotated out of the double helix, with the uracil bound in an active-site pocket. A catalytic role for base-flipping is possible via a strain mechanism, but this has not yet been established. UDG's show strong sequence conservation for an active site His/Asp diad, which are (sic) likely to be involved in catalysis. Interestingly, the MUG enzyme appears to lack these residues or other identifiable catalytic residues. Details of the mechanism relating to the possible function of active site residues and base-flipping are the target of this proposal. Kinetic studies which feature V/K kinetic isotope effects (KIE's) and equilibrium binding isotope effects (EBIE's) will be used to deduce and compare the mechanisms of the UDG's and MUG. A series of oligonucleotide substrates containing 2'-deoxyuridine with isotopic labels will be synthesized and used for KIE studies. Ground state and transition state C-glycoside analogs will be synthesized and evaluated as inhibitors. The inhibitors will be prepared in deuterated form, incorporated into oligonucleotides, and used to measure EBIE's. The results of the KIE and EBIE experimental data will be used to analyze both transition state structure and identify forces acting on the bound substrate over the reaction coordinate. These analyses will be guided by a combined ab-initio and empirical computational approach. The combined use of KIE's and EBIE's as developed here will fill a gap in the knowledge base for DNA glycosylase mechanisms, and may provide a novel way to distinguish ground state and transition state binding effects in catalysis.
