E. coli MutY and its human homologue (MUTYH, formerly MYH) play an important role in the prevention of mutations associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) by removal of misincorporated adenine residues from OG:A mismatches. This glycosylase has garnered the spotlight by 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 (referred to a MUTYH-associated polyposis or MAP). The understanding of this predisposition mechanism is still at an early stage. The clinical data is emerging rapidly, and new mutations in MUTYH continue to be uncovered. However, at this point, it is not clear whether all of the MUTYH variations affect the adenine glycosylase activity, or result in reduced OG:A repair for other reasons. We propose to further our understanding of the relationship between MUTYH and colorectal cancer, as well as continue our understanding of the complex features of mismatch recognition by this unique base-excision repair glycosylase. 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. Specifically, we will develop cellular assays to reveal features that affect recognition and repair of OG:A mismatches by MUTYH. This will involve developing several assays to assess the repair of OG:A mismatches of MutY and MUTYH in E. coli and mammalian cell lines. We will also examine the effects of alterations of the damaged substrate on MUTYH-mediated repair. In addition, we will gauge the response of these cell based assays by evaluating specific catalytic and binding defects in MUTYH. We will also examine the importance of post-translational and interactions with protein partners on the activity of MUTYH using adenine glycosylase assays, mismatch binding experiments and cellular repair assays. MUTYH expressed in baculovirus-infected insect cells is phosphorylated and we will identify the locations of these phosphorylation sites in MUTYH. We will prepare the relevent phospho-mimetic and phospho-ablating mutations in MUTYH and analyze the adenine glycosylase activity and mismatch binding affinity of the mutated enzymes We will determine the consequences of MUTYH variations found in colorectal cancer on the instrinsic enzymatic properties, ability to mediate OG:A repair and prevent mutations in cells. This will include measuring the adenine glycosylase activity, mismatch affinity, the ability to mediate OG:A repair in both bacterial and mammalian cellular assays, the genomic mutation rate and sensitivity of cells expressing MUTYH variants to oxidative stress. This information will provide information as to whether a variant MUTYH is dysfunctional, and the origin of the dysfunction.
This project will provide information on the functional consequences of variations in the DNA repair enzyme MUTYH that are correlated with the inherited colorectal cancer syndrome known as MUTYH-associated polyposis (MAP). This information will provide insight into how MUTYH variations lead to dysfunction in DNA repair resulting in the accumulation of deleterious mutations, and thereby setting the stage for colorectal cancer.
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|Banda, Douglas M; Nuñez, Nicole N; Burnside, Michael A et al. (2017) Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine. Free Radic Biol Med 107:202-215|
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|Manlove, Amelia H; McKibbin, Paige L; Doyle, Emily L et al. (2017) Structure-Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase. ACS Chem Biol 12:2335-2344|
|Wickramaratne, Susith; Banda, Douglas M; Ji, Shaofei et al. (2016) Base Excision Repair of N(6)-Deoxyadenosine Adducts of 1,3-Butadiene. Biochemistry 55:6070-6081|
|Shen, Yan; McMackin, Marissa Z; Shan, Yuxi et al. (2016) Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34. PLoS One 11:e0151026|
|Woods, Ryan D; O'Shea, Valerie L; Chu, Aurea et al. (2016) Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases. Nucleic Acids Res 44:801-10|
|Mullins, Elwood A; Shi, Rongxin; Parsons, Zachary D et al. (2015) The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions. Nature 527:254-8|
|Brinkmeyer, Megan K; David, Sheila S (2015) Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1. DNA Repair (Amst) 34:39-51|
|Engstrom, Lisa M; Brinkmeyer, Megan K; Ha, Yang et al. (2014) A zinc linchpin motif in the MUTYH glycosylase interdomain connector is required for efficient repair of DNA damage. J Am Chem Soc 136:7829-32|
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