The Enzymology of DNA Repair and MutagenesisOverall philosophy My research is based upon the belief that if we understand the mechanisms of cellular processes involved in mutagenesis and carcinogenesis we can design strategies for the prevention or treatment of cancer. My research is focused in projects elucidating the mechanisms of DNA repair, DNA replication, and mutagenesis.
My approach is to use organic synthesis and enzyme kinetics to examine the active site chemistry of the target enzymes. By this approach we are able to probe the structures of transitions states of enzymes reactions. By this approach, we expand upon the NMR and X-ray crystallography experiments, which can only examine ground state intermediates, to investigate the structures of the transition states. A project typically consist of four phases: (1) formulation of a hypothesis based upon previous biochemical studies, especially structural studies using NMR or X-ray crystallography, (2) design of a substrate, typically an unnatural substrate, to test the hypothesis,. (3) synthesis of the substrate, and (4) kinetic evaluation of activity of substrate. Reactivation of O6-alkylguanine-DNA alkyltransferase O6-Alkylguanine-DNA alkyltransferase (AGT) is a protein that repairs O6-alkylguanine residues that result from endogenous sources such as S-adenosylmethionine and environmental toxins. During the repair reaction, the alkyl group is transferred to a cysteine residue in the active site of AGT. The original guanine residue is regenerated but the cysteine remains alkylated and the protein becomes inactive. Consequently, AGT is not an enzyme but a stoichiometric reactant. Thus, an entire protein is created to repair a single damaged base. The long-term goal of this project is to create a reagent to reactivate AGT by accepting the methyl group from the S-methylcysteine. The short-term goal is to understand the mechanisms by which AGT repairs the DNA. Mechanisms of fidelity and mutagenesis of DNA polymerases. The long-term objective of this project is to understand the chemical interactions that govern the high fidelity replication of DNA. In the short-term, the goal of this research project is to test the hypothesis that specific interactions between DNA polymerases and the minor groove of DNA are crucial to the faithful replication of DNA. The interactions between the polymerase and minor groove will be studied using a combination of amino acid substitution of the polymerase and atomic substitution of the DNA. Using atomic substitution of the DNA (3-deazaguanine and 1-(3-hydroxy-4-hydroxymethylcyclopentyl)uracil shown below) and site-directed mutagenesis of E. coli DNA polymerase I we have proposed that Arg668 makes a hydrogen bonding fork with the primer terminus and incoming dNTP. These interactions can serve as a sensor for Watson-Crick geometry at these positions. | 
