AbstractDamage to DNA is ubiquitous, diverse, and ever present. Responding to DNA damage requires proper coordination among distinct repair pathways. Specialized DNA repair pathways have evolved to combat specific types of DNA damage, and their significance is underscored by the conservation of these pathways. Communication breakdowns within and between the pathways can escalate into more deleterious consequences for genomic integrity. One such pathway, Base Excision Repair (BER), has evolved to target specific lesions, most commonly small non-helix distorting nucleobase lesions. These lesions maintain a high chemical and structural similarity with canonical nucleobases and as such can well hide in plain sight. Identification, differentiation, and excision of these lesions is the primary role for a class of enzymes referred to as DNA glycosylase.
The BER glycosylase MUTYH has the unique role of recognizing the post-replicative lesion 8-oxo-7,8-dihydroguanine (OG). OG is an oxidized form of guanine that, upon acquiring a C-8-oxo N-7H, gains the ability to base pair with adenine (A). By removing misinserted adenines, MUTYH cooperates with Human Oxoguanine Glycosylase 1 (HOGG1) and MutT to target OG related lesions from multiple contexts, such as the nucleotide pool (dOGTP), pre-replication (OG:C base pairs), and post-replication (OG:A mismatches). Absence of MUTYH function on OG:A mispair leads to an accumulation of G:C to T:A transversion mutations. Inherited biallelic mutations in MUTYH are associated with the cancer predisposition syndrome MUTYH-associated polyposis (MAP) and contributes to an increased lifetime risk of colorectal cancer. Notably, hundreds of inherited MUTYH variants have been identified in patients since the identification of MAP. A significant gap in the biochemical and molecular characterization of the majority of clinically identified mutations exists warranting further study.
A central question in studying DNA glycosylases revolves around understanding how this class of enzymes distinguish their target substrate within vast excess of undamaged DNA. Extensive experimentation has been dedicated to fully elucidating v arious glycosylases mode of DNA scanning and searching for their target nucleobase across the genome. MutY has the additional challenge of detecting a damaged nucleobase while catalyzing N-glycosidic bond breakage of a canonical nucleobase. Therefore, mechanisms to avoid error driven catalysis is an important facet of MutY activity. Structural analysis via X-ray crystallography has suggested a short loop within the MUTYH C-terminal domain comprising residues Phe-Ser-His (FSH) to play a role in OG:A mispair detection. Indeed, biochemical, cellular, and single molecule data support the notion that the loop His residue is utilized for initial detection of OG.
A focus for this thesis is an in-depth analysis on the proximal H of the HxFSH motif across a diverse set of experiments. Curiously, mutations of this histidine to an arginine or tyrosine are human MAP variants. Results to be discussed within demonstrated this His to Arg mutation altered the binding affinity and minimized E.coli MutY activity. Additionally, in collaboration with Andrea Lee from University of Vermont, we uncovered this conserved histidine residue to be a key factor influencing MutY diffusion. Following the work conducted herein, we suspect this mutant may indeed affect multiple functions of MUTYH.
MutY catalysis involves extracting adenine from the DNA duplex through a mechanism known as nucleotide-flipping. Structural snapshots of MutY activity, captured via X-ray crystallography, have been pivotal in guiding the direction of my project centered around better understanding the intricacies of the MutY nucleotide-flipping mechanism. Analysis of multiple structures of Geobacillus stearothermophilus (Gs) MutY has revealed that Gln48 and Thr49 act as space-filling residues. Notably, these residues appear to intercalate into DNA and interact with orphaned bases when adenine has been flipped into MutY's active site. The primary objective of this project is to elucidate the importance of the interactions that validate the presence of OG and prepare MutY for catalysis. To achieve this, glycosylase activity assays and fluorescence-based experiments were employed to investigate key components of MutY’s base-flipping mechanism. The data suggests that MutY relies heavily on a robust interaction network surrounding both OG and adenine to ensure accurate base removal in the appropriate context.
Herein, the FSH loop portion of the HxFSH motif will also serve as a case study for identifying and studying enzymatic variants of interest. To achieve this, a cell-based functional assay will be used to examine the repair of an OG:A mispair embedded in the green fluorescent protein (GFP) gene followed by variant analysis via processing of Next Generation Sequencing, NGS, data. This method will allow us to assess the functionality of various MutY variants at the FSH loop. We anticipate the results will expand our understanding and appreciation of the FSH loop significance in conferring MutY specificity on OG:A mispairs.
In summation, this thesis highlights the importance of protein-DNA interactions and the network of interactions required for proper DNA repair and therefore maintenance of genomic integrity. The increasing accessibility of sequencing creates a demand for functional data regarding MUTYH variants, and thus characterizing MAP variants in a variety of experimental approaches to determine their impact on activity is essential. Research projects within this thesis utilize mutagenesis, biochemistry, and cellular-based assays to uncover how the HxFSH motif and space filling loop enable effective and specific targeting of OG:A mispairs by MutY.
