Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2023)

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.

    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2024)
    The base excision repair glycosylase MUTYH prevents mutations associated with the oxidatively damaged base, 8-oxo-7,8-dihydroguanine (OG), by removing undamaged misincorporated adenines from OG:A mispairs. Defects in OG:A repair in individuals with inherited MUTYH variants are correlated with the colorectal cancer predisposition syndrome known as MUTYH-associated polyposis (MAP). Herein, we reveal key structural features of OG required for efficient repair by human MUTYH using structure-activity relationships (SAR). We developed a GFP-based plasmid reporter assay to define SAR with synthetically generated OG analogs in human cell lines. Cellular repair results were compared with kinetic parameters measured by adenine glycosylase assays in vitro. Our results show substrates lacking the 2-amino group of OG, such as 8OI:A (8OI = 8-oxoinosine), are not repaired in cells, despite being excellent substrates in in vitro adenine glycosylase assays, new evidence that the search and detection steps are critical factors in cellular MUTYH repair functionality. Surprisingly, modification of the O8/N7H of OG, which is the distinguishing feature of OG relative to G, was tolerated in both MUTYH-mediated cellular repair and in vitro adenine glycosylase activity. The lack of sensitivity to alterations at the O8/N7H in the SAR of MUTYH substrates is distinct from previous work with bacterial MutY, indicating that the human enzyme is much less stringent in its lesion verification. Our results imply that the human protein relies almost exclusively on detection of the unique major groove position of the 2-amino group of OG within OGsyn:Aanti mispairs to select contextually incorrect adenines for excision and thereby thwart mutagenesis. These results predict that MUTYH variants that exhibit deficiencies in OG:A detection will be severely compromised in a cellular setting. Moreover, the reliance of MUTYH on the interaction with the OG 2-amino group suggests that disrupting this interaction with small molecules may provide a strategy to develop potent and selective MUTYH inhibitors.
      Cover page: Cellular Repair of Synthetic Analogs of Oxidative DNA Damage Reveals a Key Structure–Activity Relationship of the Cancer-Associated MUTYH DNA Repair Glycosylase
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2022)
      A-to-I RNA editing is widespread in human cells but is uncommon in the coding regions of proteins outside the nervous system. An unusual target for recoding by the adenosine deaminase ADAR1 is the pre-mRNA of the base excision DNA repair enzyme NEIL1 that results in the conversion of a lysine (K) to arginine (R) within the lesion recognition loop and alters substrate specificity. Differences in base removal by unedited (UE, K242) vs edited (Ed, R242) NEIL1 were evaluated using a series of oxidatively modified DNA bases to provide insight into the chemical and structural features of the lesion base that impact isoform-specific repair. We find that UE NEIL1 exhibits higher activity than Ed NEIL1 toward the removal of oxidized pyrimidines, such as thymine glycol, uracil glycol, 5-hydroxyuracil, and 5-hydroxymethyluracil. Gas-phase calculations indicate that the relative rates in excision track with the more stable lactim tautomer and the proton affinity of N3 of the base lesion. These trends support the contribution of tautomerization and N3 protonation in NEIL1 excision catalysis of these pyrimidine base lesions. Structurally similar but distinct substrate lesions, 5-hydroxycytosine and guanidinohydantoin, are more efficiently removed by the Ed NEIL1 isoform, consistent with the inherent differences in tautomerization, proton affinities, and lability. We also observed biphasic kinetic profiles and lack of complete base removal with specific combinations of the lesion and NEIL1 isoform, suggestive of multiple lesion binding modes. The complexity of NEIL1 isoform activity implies multiple roles for NEIL1 in safeguarding accurate repair and as an epigenetic regulator.
        Cover page: NEIL1 Recoding due to RNA Editing Impacts Lesion-Specific Recognition and Excision
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2020)
        Direct measurement of DNA repair enzyme activities is important both for the basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Herein, we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change. This strategy utilizes glycosylase-induced excimer formation of pyrenes, and modified DNA probes, incorporating two pyrene deoxynucleotides and a damaged base, enable the direct, real-time detection of NTH1 activity in vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to the identification of a new small-molecule inhibitor with sub-micromolar potency.
          Cover page: An Excimer Clamp for Measuring Damaged‐Base Excision by the DNA Repair Enzyme NTH1
          Top