G/A MISMATCH RECOGNITION AND REPAIR BY E COLI MUTY

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Mismatch repair has recently attracted a great deal of attention due to the discovery that mutations in mismatch repair enzymes are linked to hereditary colon cancer. E. Coli has an elaborate line of defense against the deleterious effects of mismatches. Decades of work on the E. Coli systems has significantly advanced the understanding of these important enzymes. However, even with years of research on the bacterial enzymes, many important and unanswered questions concerning DNA repair enzymes remain. Indeed, the factors influencing the recognition of DNA damage including the effects of sequence environment surrounding the damage are still unclear. The E. Coli enzyme, MutY is important in the prevention of mutations caused by mismatches that arise due to miscoding from oxidative damage to DNA. MutY acts as an adenine glycosylase to remove an undamaged adenine paired with 7,8-dihydro-8-oxoguanine (OG). Very little mechanistic work has focused on glycosylases that act on purine bases. An unusual aspect of MutY is that it is a [4Fe-4S] cluster-containing protein and has significant homology to the DNA repair enzyme, endonuclease III. The possibility that the [4Fe-4S] cluster domain could represent a new motif for mismatch recognition and repair is indeed quite intriguing. Thus, MutY is not only functionally important in maintaining high fidelity DNA replication, it also has unique structural and mechanistic properties. The goal of the proposed research is to provide a detailed understanding of the structural and functional properties of MutY. The initial focus will be determining the properties of G:A and OG:A mismatch containing substrates which are critical for recognition and repair by MutY. In particular, the influence of the sequence environment around the G:A mismatch will be determined as well as the a determination of the origin of sequence dependent effects. Modified G:A and OG:A containing substrates will be prepared to determine the important functional groups on the G/OG or the A that are required for efficient mismatch recognition. Novel inhibitors for MutY will be prepared to provide mechanistic information and as tools for biochemical and spectroscopic studies of the enzyme- inhibitors complex. A solid understanding of the properties of the DNA substrates for MutY will provide the subsequent basis for investigation of the properties of MutY-DNA complexes with a particular emphasis on elucidation of the role of the unusual metal center in the enzyme. These experiments will focus on determining whether or not the [4Fe-4S] cluster is intimately involved in mismatch recognition and DNA binding.