The MUTYH glycosylase has garnered the spotlight with the discovery of a correlation between inherited MUTYH variations and colorectal cancer (CRC). Using pre-steady kinetics and noncleavable 2'- deoxyadenosine analogue-containing oligonucleotides, we provided information 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 (now referred to a MUTYH-associated polyposis or MAP). 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 aberrant functioning MUTYH leads to the accumulation of mutations in tumor suppressor genes, like APC. Since the original discovery of MAP, over 100 different variants of MUTYH have been discovered. Our laboratory has 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 colorectal cancer. We have recently developed mammalian cell assays that we will use to evaluate the repair of modified substrates in cells. This will allow us to gauge how catalytic and binding defects impact overall repair in cells. We have recent results with the bacterial enzymes that suggests that the 2-amino- group of OG is critical for initial localization f OG, and we will further test this hypothesis by making additional derivatives and directly monitoring the search process using single-molecule techniques. This will provide important information into features that are involved in the initial damage detection step, and allow for better prediction of features that made compromise damage detection. We also will use our enzymatic assays in conjunction with cellular assays to provide insight into the role of the interdomain connector (IDC) region, and the Zn(II) coordination site we recently discovered on mediating efficient repair, and coordinating with downstream repair proteins, and proteins involved in damage signaling. Lastly, we also will elaborate on work that implicated MUTYH activity in increasing the sensitivity of cells to alkylation damage. As part of this work, we will develop novel methods to tag sites of MutY glycosylase activity in cells. The significance of this work is that it will provide important information as to the how MUTYH dysfunction, either inherited or acquired, may set the stage for carcinogenesis. In addition, the role of MUTYH in mediating cellular responses to oxidative damage and alkylation may provide new strategies to target cancer cells.