UC Davis

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.