RECOGNITION AND REPAIR OF MISMATCHED DNA BY MUTY

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DNA repair has attracted considerable attention due to increasing number of examples linking dysfunctional DNA repair with cancer. In particular, oxidative damage to guanine is emerging as an important causative event leading to mutagenesis and carcinogenesis. Indeed, complex repair pathways for the repair of mutations caused by 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) have been uncovered. MutY plays an important role in the prevention of mutations by removal of misincorporated adenine residues from OG:A mismatches. MutY belongs to a superfamily of base-excision repair DNA glycosylases and contains a a [4Fe-4S] cluster which is more commonly found in electron transfer proteins. Thus, MutY is not only functionally important in maintaining high fidelity DNA replication, it also has unique structural and mechanistic properties. This laboratory has developed a multifaceted research program aimed at providing a detailed understanding of the structural and functional properties of MutY. Our program has initially focused on investigating the basic properties of MutY and clarifying important issues of substrate specificity and chemistry catalyzed by MutY. In addition, we have prepared novel noncleavable substrate analogs for MutY that are effective substrate mimics to characteize the MutY-substrate DNA complex. With this solid understanding of the basic enzymatic properties of MutY, we will turn our attention to investigating in greater detail the properties of MutY involved in damaged DNA recognition and repair. Four specific areas will be examined: (1) We will further characterize the intrinsic chemistry of MutY's recognition and repair of G:A and OG:A mismatches. (2) We will determine the intrinsic properties of G:A and OG:A mismatches that are involved in their recognition and repair by MutY by making specific functional group substitutions on the mismatch or by altering the sequence surrounding the mismatch to determine the recognition elements required by MutY. (3) We will investigate the properties of the MutY-DNA complex using structural and biochemical methods to gain insight into factors involved in damage specific recognition by DNA repair enzymes. (4) We will investigate the role of MutY's [4Fe-4S] cluster loop motif (FCL) in damaged DNA recognition by making specific alterations in amino acids of the FCL and determining the effects on MutY's functional properties.