UC Davis

DNA repair is a highly coordinated process that requires proper activation and repression of specific proteins and cofactors. Without proper coordination of the players involved, DNA repair can quickly progress into more detrimental consequences for genomic integrity. Specialized DNA repair pathways have evolved to combat specific types of DNA damage, and their significance is underscored by the conservation of these pathways. The base excision repair (BER) pathway is one such pathway that has developed to target specific lesions, most commonly those that are non-helix distorting. These lesions can be difficult to identify due to their tendency to minimally perturb the helix and hide in plain sight.The BER glycosylase MUTYH is uniquely poised to recognize the post-replicative lesion 8-oxo-7,8-dihydroguanine (OG) paired with adenine (A). The failure of MUTYH to properly recognize the OG:A mispair and excise the undamaged adenine would allow for a transversion mutation to be sealed into the DNA. Indeed, inherited biallelic mutations in MUTYH can cause the cancer pre-disposition syndrome called MUTYH-associated polyposis (MAP) and contributes to an increased lifetime risk of colorectal cancer. The founding MAP mutations are Tyr179Cys and Gly396Asp, but over 300 mutants have been identified in patients since the identification of MAP in 2002. The founding variants have been extensively characterized, but there is a significant gap in the biochemical and molecular characterization of the majority of these clinically identified mutations. This dissertation examines the consequences of MAP variants and their effects on the roles of MUTYH in repair and protein-protein interactions with special consideration to the interdomain connector (IDC). Specifically, this dissertation examines a subset of MAP variants within the IDC, which is well characterized as a flexible and dynamic region that is involved in multiple protein-protein interactions that are required for downstream DNA repair signaling. Initial in vitro characterization of these variants in the IDC did not reveal notable differences between these mutants and the wild-type protein. However, evaluation using a newly optimized mammalian cell assay revealed that these mutations indeed reduce OG:A repair in a cellular context. This study underscores the importance of a more holistic characterization of MUTYH function and its roles rather than a targeted approach as done using in vitro experiments. These cellular repair assays can then inform additional studies by revealing whether an overall deficiency in OG:A repair is observed. Mutations in dynamic regions of MUTYH may impact more than one function of the protein. The interactions of these MAP variants are discussed in the context of BER progression specific to the MUTYH interaction with the next key player in OG:A repair, AP endonuclease 1 (APE1), as these mutations reside in or adjacent to the putative binding domain of MUTYH and APE1. Additionally, a novel protein partner of MUTYH , UV-damaged DNA-binding protein complex (UV-DDB), is identified that may aid in the repair of OG:A mispairs by making them more accessible for repair. UV-DDB has recently been identified to bind to OG containing base pairs and aid in turnover of both enzymes involved in their repair. This interaction with UV- DDB may be integral to MUTYH activity in the presence of DNA packing and nucleosomes. Lastly, additional studies of MUTYH are described to determine whether MUTYH may be responsive to oxidative stress and reactive oxygen species (ROS) within the cell. These species are known to damage macromolecules within the cell, including DNA and proteins. Thus, damage to MUTYH by ROS may be a mechanism by which MUTYH responds to different levels of oxidative stress and regulate its activity. In collaboration with the Weerapana group at Boston College, we identify one cysteine residue as highly reactive and propose this reactivity may be key to this regulatory mechanism. Curiously, mutation of this cysteine to arginine corresponds to a MAP variant in the IDC and identified as possessing reduced repair. This mutant may indeed influence multiple roles of MUTYH due to its location and roles within the protein. Taken together, this dissertation highlights the importance of protein-protein interactions and the networks required for proper DNA repair and genomic maintenance. DNA repair is a team sport and requires the support of many proteins. It is essential to examine MAP variants in more than one way to determine how these variants may impact OG:A repair and roles of MUTYH in preserving genomic integrity. The increasing accessibility of sequencing creates a similar increase in demand for information regarding mutations in MUTYH, and this dissertation supports the understanding of how MUTYH functions in a more biologically relevant manner.