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.

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.

The genomic information of all living organisms is susceptible to alterations through chemical modifications in DNA occurring from both environmental and cellular sources. To prevent the detrimental effects of DNA damage, DNA repair pathways were acquired and evolved to target and repair certain modifications in DNA. Guanine Oxidation (GO) repair pathway is one of the several pathways that uses DNA glycosylases to target DNA damage resulting from oxidation of guanine. The adenine glycosylase MutY is one of the key players in this pathway that removes the undamaged adenine (A) base across an oxidation product of guanine (G), 8-oxo- guanine (OG).

The focus of this work is to understand the mechanistic and structural details of how MutY locates and differentiates its target mispair from other similar base pairs and excises the misincorporated adenine. The significance of MutY in maintaining the integrity of DNA is highlighted by its relevance to inherited colorectal cancer syndrome known as MUTYH-associated polyposis (MAP). Previous studies revealed the importance of the C-terminal domain (CTD) of the protein in OG recognition and the roles of the residues in the active site of N-terminal domain (NTD) in adenine excision mechanism. The goal of my thesis work is to elucidate roles of residues involved in OG recognition and adenine excision mechanisms by using enzymology, nucleic acid chemistry and X-ray crystallography.

This work reveals the role of a CTD loop comprising Phe, Ser and His residues, referred as FSH loop, that presumably involved in OG recognition provided by structural implications. Our structural and biochemical results showed the significance of FSH loop within the CTD, in OG versus G recognition. Indeed, the replacement of more than one residue in the FSH loop results in loss of specificity for OG. Furthermore, the replacement of serine in the loop affected the overall activity of MutY, albeit showing similar specificity as the wild-type enzyme. We believe the reduced activity with FAH is due to the involvement of serine residue in a hydrogen bond network around OG. The residues in this hydrogen bond network may all work in conjunction to ensure faithful OG recognition and adenine placement in the active site.

The OG lesion is known to pair with adenine in syn conformation in duplex DNA, however OG is found in anti conformation in the crystal structures of MutY bound to DNA. The hydrogen bond network around OG in the anti conformation involves contacts with serine of FSH loop and two NTD Gln and Thr residues. The Gln and Thr residues from NTD intercalates into the space left from extruded adenine. We explored the roles of these intercalation residues by mutational analysis and structure activity relationship (SAR) studies with a series of previously studied OG analogs. The kinetic and binding results indicated Gln and Thr residues may have a role in OGanti recognition and in proper placement of adenine in the active site for removal.

The recent structural and biochemical studies from David and Horvath Laboratories led to a proposed double replacement mechanism for MutY. In this mechanism, a highly conserved catalytic Asp residue is proposed to form a covalent intermediate. We replaced the Asn residue that is the hydrogen partner of catalytic Asp to study the additional features of the adenine excision mechanism. The structural and kinetics results of Asn146Ser variant indicated that the loss of hydrogen bonding to Asp resulted in significantly impaired activity and an increase in the pKa of Asp. Next, we obtained a crystal structure of N146S in complex with a an alternative substrate OG:Purine (P). To our surprise, we captured the OG:P substrate before N-glycosidic bond hydrolysis occurred in the active site. In addition, a calcium ion coordinated to the base and catalytic Asp was found in the space previously occupied by Asn side chain that is likely to be responsible for the inhibition of the base removal. The further crystallization trials with extended incubation time or removal of calcium afforded the first structures of Asn146Ser variant in complex with enzyme-catalyzed β-anomer AP site formation consistent with retaining mechanism. The combination of the crystal structures forms a structural movie of base excision mechanism by N146S variant that provides further insight into the MutY adenine excision mechanism.

In the final chapter, I describe a computational study on MutY structures in complex with either a substrate or transition state analog to identify the hot spots of the enzyme for ligand binding. These models and computational results showed an Arg residue may either open or close the end of the active site channel. We further investigated the role of Arg by replacing it with a Gly or Trp residue to either open or close the channel, respectively. Our biochemical findings suggested Arg may be indirectly affecting the adenine excision and this region may be targeted as an allosteric site for drug discovery.

Taken all together, this work sheds light into the roles of residues involved in OG recognition, adenine positioning and excision in the active site. Furthermore, we identified the FSH loop and adenine exit channel as allosteric sites that can affect the adenine excision activity indirectly. The development of small molecule inhibitors targeting these regions may provide the basis for novel cancer chemotherapeutics.

Deoxyribonucleic acid (DNA) is susceptible to DNA damage and one form of DNA damage is caused by reactive oxygen and nitrogen species (RONS). RONS can oxidize guanine (G) into 8-oxo-7,8-dihydro-2’-deoxyguanosine (OG), which on its Hoogsteen face mimics thymine. During replication, adenine (A) can be placed across from OG resulting in a transversion mutation. To prevent the accumulation of mutations, the GO repair pathway has base excision repair enzymes to either cleave out OG across from cytosine (C) or A from OG:A base pairs. The enzyme responsible for the removal of A from OG:A base pairs is MutY in bacteria and Homo sapiens MutY, MUTYH, in humans. MUTYH-associated polyposis (MAP) is a genetic disease that results in early onset of colorectal cancer (CRC) due to inheritance of MUTYH variants. The decreased activity and function of MUTYH variants leads to overabundance of somatic mutations and inactivation of tumor suppressor genes, leading to CRC.This thesis reviews two different MutY enzymes, the first is Geobacillus stearothermophilus (Gs) MutY to determine the effects of cancer variant R241Q and the second is Lactobacillus brevis MutY (LbY) to begin to understand the nature of this “clusterless” MutY. Both enzymes will be reviewed mechanistically and structurally. MutY has several motifs of interest including the iron-sulfur (FeS) cluster and the FSH (Phe-Ser-His) loop. The FeS cluster is coordinated by four highly conserved cysteine (Cys) residues. Neighboring these four Cys are four arginine residues (Arg) in which previous research proposes that these Arg hydrogen bond with the conserved Cys coordinating the FeS cluster. To understand if alterations to one of these Arg could affect enzymatic activity and binding, cancer databases were searched for related mutations for all four of the Arg. Several mutations were found for each corresponding Arg. The variant studied in this thesis was found in a patient with MAP and breast cancer, R241Q. Preliminary unpublished A glycosylase assays demonstrate binding of R241Q but no activity. Enzymatic studies of other MAP variants have been done previously in Gs MutY, so the corresponding mutation, R149Q, was tested and was found to have activity. Previous research demonstrates the importance of the FeS cluster in DNA repair and recognition, however LbY does not contain the FeS cluster coining it as a “clusterless” MutY. Also, LbY does not have a serine (Ser) in the FSH loop but rather a threonine (Thr). In the FSH loop of Gs MutY, Ser308 hydrogen bonds with OG, but the structural differences between the hydroxymethyl of Ser and the secondary hydroxyl group of Thr could mean that LbY recognizes OG differently than Gs MutY. The goal of this thesis is separated into two parts: first, to determine the impact of the Arg149 to Gln149 mutation in Gs MutY to the hydrogen bond network, the active site, and the FeS cluster and second, to optimize the purification of LbY for structural studies as well as beginning to understand the role of Thr in the FTH loop. In this thesis, R149Q Gs MutY was successfully crystallized with DNA duplexes containing OG opposite of tetrahydrofuran (THF), a product analog, and purine (P), a substrate analog. The biochemical results demonstrated decreased activity in R149Q compared to wild-type (WT) Gs MutY and even lower activity with OG:P. This suggested the potential for a product structure with R149Q OG:P and the product structure was captured. The best fit sugar pucker conformation of the product was 3’-exo-beta, which is found to be the most stable conformer of the oxocarbenium ion intermediate during A cleavage. Analysis of both crystal structures found that the effects of Arg mutation to Gln are dramatic: there are alternate conformations of the FeS cluster and the supporting Cys, a calcium ion is found coordinating with Asn146, and Gln149 can no longer coordinate the DNA backbone. Due to the shorter side chain of Gln149, a Ca2+ ion fills that space and coordinates Asn146. Asn146 forms hydrogen bonds with Asp144, so to maintain these connections Asp144 was found further away from the substrate in both R149Q-OG:AP, the product structure, and R149Q-OG:THF compared to the Transition State Analog Complex structure. These results illustrate the potential supportive role of these Arg residues near the coordinating Cys of the FeS cluster. LbY was successfully purified in this thesis, but crystals could not be obtained. Further crystal conditions will need to be tested for LbY. Previous research has demonstrated the importance of the FSH loop by amino acid substitution studies, which resulted in decreased MutY activity. Previous research found that Ser hydrogen bonds with N7 and O8 of OG, however due to the different side chains of Ser and Thr, Thr may not be able to maintain these interactions with OG. To begin to understand OG recognition in LbY MutY, biochemical experiments were conducted with OG analogs, 8-thioguanine (8SG), 8-bromoguanine (8BrG), and 7-methyl-8-oxo-7,8-dihydroguanosine (7MOG), to see how MutY activity is affected by changes to N7 and O8 positions of OG. The OG analogs, G, 8SG, and 8BrG have modifications to the O8 position, but 8SG had the fastest rate of glycosylase bond cleavage of these three. G and 8BrG had similar rates of glycosylase bond cleavage which could be due to the modifications of the O8 position. 7MOG had the lowest rate in activity which could be due to the bulkiness of the methyl group and potential steric interactions with Thr316. The size of 8SG and 8BrG could also affect substrate recognition due to the size of the substrate and the side chain of Thr316. This thesis furthers our understanding of the FeS cluster, expanding it beyond the coordinating Cys to the potential Arg near Cys, and illustrates the potential differences in OG recognition due to the FTH loop. Further research could investigate the other Arg residues in this area to see if they have similar effects to the active site and the FeS cluster. Additionally, continued attempts to crystalize LbY for structural analysis will increase the understanding of the FeS cluster area and the FTH/FSH loop comparison.

The DNA nucleobases are highly susceptible to modification from a wide variety of oxidizing and alkylating agents. Alterations to the nucleobase can threaten genomic integrity by disrupting normal cellular processes including replication and transcription or by introducing mutations. Damage to the DNA base is identified and removed by DNA glycosylases that initiate the base excision repair pathway (BER). The diversity of DNA damage, the location of damage, and how that DNA damage is identified has given valuable insights into disease related to DNA repair. The NEIL family of DNA glycosylases can excise lesions arising from all four nucleobases in a variety of DNA contexts, including duplex, single stranded, and G quadruplex (G4). The loss or dysfunction of NEIL enzymes is associated with a diverse set of disease phenotypes, such as immune deficiencies, anxiety, impaired memory retention, and cancer. Yet, it remains unclear how NEIL dysfunction is related to these phenotypes. In this work, I evaluate the molecular and structural features involved in recognition and excision of a diverse set of lesions across multiple DNA contexts by the NEIL family of glycosylases. First, I examine the features of the damaged nucleobase that influence differences in excision between the two isoforms of NEIL1. RNA editing of the NEIL1 pre-mRNA by the Adenosine Deaminase Acting on RNA (ADAR1) creates two isoforms of NEIL1 via a recoding event that converts a lysine to arginine in the lesion recognition loop of NEIL1. Notably, previous studies have demonstrated that the two isoforms display different enzymatic properties on the pyrimidine lesion, thymine glycol (Tg), where the unedited (UE, K242) isoform showed a significantly faster rate of excision compared to edited NEIL1 (Ed, R242). To further the understanding of NEIL1 activity, I continued this analysis on lesion processing by the two NEIL1 isoforms on a large number of substrates to evaluate the impact of the ADAR1-mediated recoding event. Notably, UE NEIL1 demonstrates better excision of U/T pyrimidines, such as Tg, uracil glycol (Ug), 5-hydroxyuracil (5-OHU), and 5-hydroxymethyluracil (5-hmU), than the edited isoform. However, the relative difference in the rate of excision between Ed and UE NEIL1 is not consistent between lesions and decreases markedly in the series with Tg > Ug > 5-OHU > 5 hmU. Calculations performed in the gas phase examine tautomer stability (2-OH vs 4-OH) and N3 proton affinity of each lesion, and the relative rates of base excision track with the N3 proton affinity of the most stable tautomer. These data suggest that enzyme-promoted tautomerization affects cleavage of the glycosidic bond and enhances excision observed with the unedited enzyme. As a result, the differences in activity between the two isoforms of NEIL1 imply a unique regulatory mechanism for DNA repair by RNA editing. Additionally, the ability of NEIL enzymes to excise DNA damage from G4 structures was evaluated. The G-rich nature of G4 sequences makes them highly susceptible to oxidative damage, and DNA damage in promoter containing G4 sequences and interaction of BER enzymes have been implicated in gene regulation. An oxidation product of guanine, guanidinohydantoin (Gh), was positioned at varying locations in G4 sequences from VEGF, KRAS, and RAD17 promoters, and excision by NEIL1 and mouse NEIL3 (mNeil3), was monitored using in vitro glycosylase assays. The production curves are biphasic providing two rates, indicating that a fraction of Gh within the G4 is excised rapidly, while another fraction is processed more slowly. Also, the percent base removed does not reach 100% despite excess enzyme. The G4 sequence was the largest contributing factor to the differences in rates, associated amplitudes, and overall Gh excision by the NEIL enzymes. These observations suggest that the G4 structure and stability impact accessibility and ability of NEIL1 and mNeil3 to position Gh for cleavage. Thus, reduced repair by NEIL may increase the persistence of mutations or alter gene regulation. In the last chapter, I evaluated the repair of Tg, Gh, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) in human cell lines, as repair can vary between them, using a green fluorescent protein (GFP) reporter that had been previously used to evaluate the repair of Gh and Tg initiated by Neil1 in MEFs. Here, I evaluated the repair of Tg, Gh, and FapyG in HEK293FT cells and the cancer cell lines, HeLa and U87. The extent of repair was the greatest in HEK 293FT cells, while reduced repair of all lesions was observed in HeLa and U87 cells. In comparing the lesions, FapyG was repaired to the greatest extent followed by Gh and Tg across all cell lines. These lesions have never been explored in a cell-based assay, and lesion containing plasmid based cellular assays are useful tool to examine DNA damage and DNA repair capacity as they reflect the influence of factors such as protein expression and stability, which can be altered in disease states. My dissertation evaluates the molecular basis of NEIL initiated repair to provide insights between NEIL activity and its diverse disease phenotypes. These results demonstrate that the type of damage and the location of damage can influence excision by the NEIL DNA glycosylases and altered NEIL activity can impact cellular function and progression of disease. NEIL1 specificity can be modified by RNA editing, and aberrant RNA editing and high levels Ed NEIL1 is associated with cancer. Also, NEIL enzymes interact with G4 structures and reduced excision may lead to increase mutation or participate in regulatory mechanisms. Each chapter examines unique repair properties of the NEIL enzymes and its DNA damage substrates and provides further opportunities to explore repair of these diverse lesions.

The chemical structure of DNA is fundamental for proper development and function of an organism’s cells and is responsible for the mechanisms of genetic inheritance and evolution. However, the chemical properties of the nucleobases make them highly susceptible to chemical modification and structural alterations that threaten genomic integrity. At the forefront of the battle against such chemical modifications are the DNA glycosylases that initiate the base excision repair pathway (BER) by cleaving erroneous nucleobases from the DNA backbone. The NEIL family of DNA glycosylases (NEIL1, 2, and 3) are unusual from other BER glycosylases in the fact that they can recognize and excise a wide range of modified nucleobases and can do so from several non-canonical DNA contexts. The loss or dysfunction of these enzymes is linked to a wide variety of diseases including metabolic and mental acuity diseases, as well as several types of cancer. Yet the direct relation between these diseases and NEIL activity is unclear. A molecular understanding of the mechanisms of substrate recognition and catalysis will be critical to better understanding their role in cellular disease pathways and to providing potential targets for therapeutic design. Using a variety of chemical biology approaches, this dissertation investigates the mechanisms of NEIL activity on a variety of substrates in multiple DNA contexts and conditions to better understand some of the unusual aspects of NEIL behavior.

In chapter two, the ability of NEIL1 and NEIL3 to remove oxidative modifications from G4 DNA in comparison to single strand DNA and canonical duplex DNA contexts was evaluated. Initial studies investigated the ability of the NEIL enzymes to remove Gh from several positions within three potentially G4 forming promoter sequences. By using in vitro glycosylase assays, we observed product production curves that were sequence and enzyme dependent, and that demonstrated base removal by two distinct rates previously observed with NEIL1 under different conditions. To further investigate these phenomena, I expanded testing to include additional lesions, FapyG, 5-OHU and an AP site, in not only the G4 context, but also single strand and duplex DNA, providing a complex matrix allowing comprehensive comparison of the in vitro kinetics. Collectively, the data showed that the base identity dictated whether removal occurred and suggested that the enzymes may be binding non-productively to the target substrates. Additional binding studies performed showcased that NEIL could bind to all DNA context equivalently despite differential repair, highlighting a potential role for NEIL-non-productive binding to prevent erroneous repair or in cellular signaling pathways related to genomic stability.

In the third chapter, the focus is on the most understudied NEIL enzyme, NEIL2, which has been profoundly under studied for its biochemical activities. To investigate the substrate scope of two mammalian NEIL2 enzymes, HsaNEIL2 and MdoNeil2, a panel of 14 different substrates in single strand and duplex DNA were tested, and both showed a distinct preference for the strand scission of AP sites. For the lesion base substrates, a structural activity relationship study revealed influences from polar or non-polar moieties around the heterocycle in a position dependent manner. We also investigated the impact of multiple NEIL2 constructs, lacking unusual, disordered regions within the enzyme, on its lyase activity. Indeed, one of these deletions significantly alters the enzymes’ ability to act on duplex DNA but not single strand DNA.

In chapter 4, the impact of macromolecular crowding on BER and the NEIL enzymes is explored. Typical in-vitro biochemical assays are run in dilute buffer, however these conditions do not effectively capture cellular conditions, where 30-40% of cellular space is taken up by large macromolecules. By mimicking the crowded environment of a cell with inert polymers, we discovered a unique pathway of regulation for NEIL1, where removal of lesions from inappropriate DNA contexts is significantly reduced. Through investigation, we revealed that this effect is specific to the glycosylase activity of NEIL1, and likely acts through an entropically driven effect involving some type of structural re-arrangement. Additionally, binding studies showed no detectible differences in binding affinity despite differential repair under crowded conditions compared to the initial uncrowded solutions. We postulate that by inducing a catalytically inactive enzyme that can still bind to substrates specifically under crowded conditions, inappropriate repair can be prevented. This showcased an unprecedented mechanism of BER activity regulation and highlights the importance of considering cellular environments when designing experiments. The final chapter seeks to perturb the catalytic roles of several structural motifs within the NEIL1 enzyme previously thought critical for activity by mutating residues involved in the catalytic mechanism. The first of these regions is known as the void filling residues, a highly conserved set of amino acids that are thought to flip the base substrate out of the helix and stabilize it within the active site. The second region is a flexible loop known as the lesion recognition loop, that harbors residues that directly contact the base substrate, and are known to have significant influence over catalytic competency. By mutating these residues to alanine, I investigated how NEIL1 catalytic activity, specifically towards Gh in single strand and duplex DNA, was altered. Several alanine mutations to the lesion recognition loop had little impact in duplex DNA, but significantly reduced the rate of removal in single strand DNA. Most surprisingly, one of the mutants with a critical residue at position 242 mutated to alanine, which our lab has previously shown to play a role in catalysis, was still highly active. In contrast several of the void filling residue mutations did slow the reaction, and mutating Met81 to Ala eliminated removal from single strand DNA.

My dissertation uses chemical-biology approaches to perturb and investigate NEIL substrate scope and specificity to provide insight to how NEIL enzymes may be behaving or misbehaving in cellular processes. Each chapter focuses on unique aspects of repair associated with the NEIL enzymes, and opens new avenues of exploration, and suggests potential mechanisms that could be targeted for therapeutic benefits.