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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2022)

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.

    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2018)
    Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease.
      Cover page: Cellular Assays for Studying the Fe–S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2020)
      The human DNA base excision repair enzyme MUTYH (MutY homolog DNA glycosylase) excises undamaged adenine that has been misincorporated opposite the oxidatively damaged 8-oxoG, preventing transversion mutations and serving as an important defense against the deleterious effects of this damage. Mutations in the MUTYH gene predispose patients to MUTYH-associated polyposis and colorectal cancer, and MUTYH expression has been documented as a biomarker for pancreatic cancer. Measuring MUTYH activity is therefore critical for evaluating and diagnosing disease states as well as for testing this enzyme as a potential therapeutic target. However, current methods for measuring MUTYH activity rely on indirect electrophoresis and radioactivity assays, which are difficult to implement in biological and clinical settings. Herein, we synthesize and identify novel fluorescent adenine derivatives that can act as direct substrates for excision by MUTYH as well as bacterial MutY. When incorporated into synthetic DNAs, the resulting fluorescently modified adenine-release turn-on (FMART) probes report on enzymatic base excision activity in real time, both in vitro and in mammalian cells and human blood. We also employ the probes to identify several promising small-molecule modulators of MUTYH by employing FMART probes for in vitro screening.
        Cover page: Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity
        • 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 (2021)
          The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4-5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.
            Cover page: Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2016)
            The sterol regulatory element-binding protein (SREBP) transcription factors have become attractive targets for pharmacological inhibition in the treatment of metabolic diseases and cancer. SREBPs are critical for the production and metabolism of lipids and cholesterol, which are essential for cellular homeostasis and cell proliferation. Fatostatin was recently discovered as a specific inhibitor of SREBP cleavage-activating protein (SCAP), which is required for SREBP activation. Fatostatin possesses antitumor properties including the inhibition of cancer cell proliferation, invasion, and migration, and it arrests cancer cells in G2/M phase. Although Fatostatin has been viewed as an antitumor agent due to its inhibition of SREBP and its effect on lipid metabolism, we show that Fatostatin's anticancer properties can also be attributed to its inhibition of cell division. We analyzed the effect of SREBP activity inhibitors including Fatostatin, PF-429242, and Betulin on the cell cycle and determined that only Fatostatin possessed antimitotic properties. Fatostatin inhibited tubulin polymerization, arrested cells in mitosis, activated the spindle assembly checkpoint, and triggered mitotic catastrophe and reduced cell viability. Thus Fatostatin's ability to inhibit SREBP activity and cell division could prove beneficial in treating aggressive types of cancers such as glioblastomas that have elevated lipid metabolism and fast proliferation rates and often develop resistance to current anticancer therapies.
              Cover page: Fatostatin Inhibits Cancer Cell Proliferation by Affecting Mitotic Microtubule Spindle Assembly and Cell Division*
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2018)
              The DNA base excision repair (BER) glycosylase MUTYH prevents DNA mutations by catalyzing adenine (A) excision from inappropriately formed 8-oxoguanine (8-oxoG):A mismatches. The importance of this mutation suppression activity in tumor suppressor genes is underscored by the association of inherited variants of MUTYH with colorectal polyposis in a hereditary colorectal cancer syndrome known as MUTYH-associated polyposis, or MAP. Many of the MAP variants encompass amino acid changes that occur at positions surrounding the two-metal cofactor-binding sites of MUTYH. One of these cofactors, found in nearly all MUTYH orthologs, is a [4Fe-4S]2+ cluster coordinated by four Cys residues located in the N-terminal catalytic domain. We recently uncovered a second functionally relevant metal cofactor site present only in higher eukaryotic MUTYH orthologs: a Zn2+ ion coordinated by three Cys residues located within the extended interdomain connector (IDC) region of MUTYH that connects the N-terminal adenine excision and C-terminal 8-oxoG recognition domains. In this work, we identified a candidate for the fourth Zn2+ coordinating ligand using a combination of bioinformatics and computational modeling. In addition, using in vitro enzyme activity assays, fluorescence polarization DNA binding assays, circular dichroism spectroscopy, and cell-based rifampicin resistance assays, the functional impact of reduced Zn2+ chelation was evaluated. Taken together, these results illustrate the critical role that the "Zn2+ linchpin motif" plays in MUTYH repair activity by providing for proper engagement of the functional domains on the 8-oxoG:A mismatch required for base excision catalysis. The functional importance of the Zn2+ linchpin also suggests that adjacent MAP variants or exposure to environmental chemicals may compromise Zn2+ coordination, and ability of MUTYH to prevent disease.
                Cover page: The Zinc Linchpin Motif in the DNA Repair Glycosylase MUTYH: Identifying the Zn2+ Ligands and Roles in Damage Recognition and Repair
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2019)
                UV-DDB, a key protein in human global nucleotide excision repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a noncanonical role in base excision repair (BER). We investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase β-gap filling activity by 30-fold. Single-molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moves to sites of 8-oxoG repair in cells, and UV-DDB depletion sensitizes cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.
                  Cover page: Damage sensor role of UV-DDB during base excision repair
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