The Role of MUTYH and NEIL in Cellular Repair of Oxidative DNA Damage
- Conlon, Savannah
- Advisor(s): David, Sheila
Abstract
DNA is under constant bombardment from a variety of different exogenous and endogenous sources that can lead to a variety of different modifications. These modifications can be especially deleterious, leading to mutagenesis, DNA replication blocks, and genomic instability. Thankfully, DNA repair pathways have evolved to prevent the effects of such DNA damage. The base excision repair pathway (BER) is responsible for the removal of oxidative DNA base damage from the genome, where a DNA glycosylase will initiate the pathway by cleaving the N-glycosidic bond between the aberrant base and the sugar. Multiple DNA glycosylases exist to remove the variety of different oxidative modifications that can arise in DNA, but in this work two are of focus. The adenine glycosylase MUTYH (MutY in E. coli) is responsible for removing the undamaged adenine base that gets misincorporated across from 8-oxo-7,8-dihydroguanine (OG), which is a common oxidative product of guanine. Indeed, the importance of MUTYH is underscored in its link to a colorectal cancer predisposition syndrome, known as MUTYH associated polyposis (MAP). The bacterial enzyme MutY has been extensively characterized in vitro and in a cellular context, but the mammalian and human protein are in the early stages of characterization. The David Laboratory has spent many years revealing the features of the adenine and OG base that the bacterial MutY relies on for recognition and repair, but we still do not yet know if the human enzyme relies on these same features. In this work, I use structure activity relationship (SAR) studies to elucidate the features of OG required for human MUTYH recognition and repair of the OG:A mispair. I use a newly designed plasmid-based reporter assay to incorporate various OG analogues, which are then transfected into WT and MUTYH -/- HEK293FT cell lines. Repair is then accessed and quantified via flow cytometry. I show that while the recognition features of the OG base remain similar to the bacterial enzyme, there are some key differences with the human enzyme that were not previously known. The NEIL DNA glycosylases are a unique family of enzymes that are capable of removing over fifteen various oxidative modifications from DNA. Additionally, these enzymes have been shown to remove these modifications from different DNA contexts, including double-stranded DNA, single-stranded DNA, bubble and bulge DNA, R-loops, and G-quadruplex structures. In this work, I aim to further reveal the processing capabilities of these enzymes. I first reveal the ability of NEIL1 and NEIL3 to remove oxidative DNA damage from a variety of different G-quadruplex structures, which are structures that have been implicated in gene regulation. We show that the NEILs are promiscuous in their processing ability from different structures and from different positions of the G-quadruplex, but that excision does not reach 100% completion. I also demonstrate binding by the enzymes to the G-quadruplex structures. Our results suggest that NEIL could be binding to these structures, but not excising the damage, suggesting a role of these base excision repair enzymes in gene regulation. I next investigate the role of the NEIL enzymes in their ability to remove 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from a variety of different DNA contexts, which has been difficult to extensively characterize due to its difficult synthesis. I show that NEIL1 and NEIL3 can remove FapyG but that guanidinohydantoin (Gh) remains its superior substrate. Lastly, I design a plasmid-based reporter assay that reports on the ability of the NEIL family of enzymes to repair 5-hydroxyuracil (5-OHU), Gh, or thymine glycol (Tg) via flow cytometry in human cells. Using NEIL1, NEIL2, and NEIL3 knockout HEK293FT cell lines, I performed a comparative repair analysis of the various DNA base modifications by NEIL. I demonstrate that even in the absence of one NEIL enzyme, all three lesions still exhibit robust repair, suggesting that all three NEIL enzymes coordinate in the repair of oxidative DNA damage. Overall, the work presented herein showcases the repair capabilities of two DNA glycosylases, MUTYH and NEIL, working to preserve genomic integrity.