MECHANISM OF DNA REPAIR ENZYMES

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Because DNA polymerase beta (Beta-pol) is responsible for gap-filling synthesis in some mammalian DNA repair pathways, it is one of the most important enzymes for maintaining the integrity of genomic DNA. B-pol is a potential target for drug design to either enhance or block the DNA repair process. However, our understanding of the molecular mechanisms of B-pol is in its infancy, so that we do not yet know enough to develop a program of rational drug design. To correct this deficiency, and to understand basic principles of the nucleotidyltransferase reaction of DNA polymerases, we will focus on a key step in the B-pol DNA synthesis mechanism, enzyme-template.primer binding. The project exploits recombinant expression of B-pol and its constituent domains in E. coli and involves: 1) Studies of the molecular structure of B-pol both by X- ray crystallography and by multidimensional NMR spectroscopy. B-pol and its domain fragments will be crystallized as complexes with the synthetic primer d(T) and with other synthetic template primers. NMR analysis will be with B-pol fragments ranging from -6 to 12- kDa, representing folded protease-resistant domains in the intact protein. Structures obtained by these approaches will be examined for implications on function by molecular modeling and site-directed mutagenesis, followed by functional assays of mutant proteins; 2) Studies of B-pol functions, such as binding to template primer and primer substrates: These will use equilibrium binding and enzymological techniques, including pre-steady kinetics. The enzyme-template.primer binding pocket, localized by photochemical cross- linking and structural studies, will be altered by site-directed mutagenesis. Studies of replication by B-pol will seek the cause of sequence variability among products of DNA synthesis by B-pol. Frameshaft mutational hot spots account for much of the variability, a process probably due to mistakes that are initiated by template.primer slippage mechanisms. We will study mutations produced in vitro to determine if template.primer-B-pol interactions play a role in the template.primer slippage. One goal of drug design targeted to B-pol is to develop agents that can potentiate chemotherapy by inhibiting DNA repair. Since gap- filling synthesis is required during repair of many types of DNA lesions, B-pol is a logical choice for drug intervention. A second goal of drug design is enhancing DNA repair by finding agents that increase the activity and/or accuracy of B-pol.