MUTYH-associated polyposis (MAP) is an inherited colorectal cancer (CRC) syndrome that results from inherited biallelic mutations in the gene for the MUTYH base excision repair (BER) glycosylase. Our laboratory provided enzymatic analysis on the functional defects of two missense variants of MUTYH found in colorectal cancer that was key for establishing the connection between MUTYH variants and colorectal cancer. MUTYH prevents mutations associated with 8-oxo-7,8-dihydro-2’-deoxyguanosine (OG) by removal of misincorporated adenine residues from OG:A mismatches, and thus aberrantly functioning MUTYH leads to the accumulation of mutations in tumor suppressor genes. Since the original discovery of MAP over 300 different mutations have been reported in MUTYH. This project is focused on revealing fundamental features of MUTYH and MUTYH variants. Our underlying hypothesis is that a detailed molecular understanding of OG:A mismatch recognition goes hand-in-hand with revealing intricacies of the relationship between MUTYH and cancer. By corrrelating results from enzymatic assays with cellular assays in E. coli and mammalian cells we have uncovered the impact of specific molecular defects in damage recognition and base excision on overall MUTYH-mediated repair. This has led to uncovering many new surprising features of MUTYH-mediated repair that will be the focus of this renewal grant application. Specifically, we will use a multi-faceted approach involving nucleic acid chemistry, enzymology, single-molecule experiments and cellular assays to analyze a large number of MUTYH variants localized near the two metal cofactors and FSH OG recognition motif to reveal insight into critical features impacting damage recognition and repair. We also will evaluate the impact of MUTYH variants in the context of chromatin. We will also evaluate how damage recognition by MUTYH in chromatin may be facilitated by the damage sensor UV-DDB. We recently showed that MUTYH binding to abasic sites in DNA mediates a cytotoxic response to alkylation damage. We will determine if the interaction of MUTYH with abasic sites is the underlying origin of the growing implication of MUTYH involvement in the response to DNA damage beyond OG:A mispairs, such as UV light and chemotherapeutic DNA damaging agents. The significance of this work is that it will provide important information into the mechanisms by which MUTYH dysfunction, either inherited or acquired, sets the stage for carcinogenesis and this information will directly influence appropriate patient management. In addition, the role of MUTYH and MUTYH variants in mediating celllular responses to oxidative damage, alkylation and UV light may provide the basis for innovative new therapeutic approaches.