DNA polymerase epsilon (Pol ε) is a multi-subunit B-family DNA polymerase that is involved in leading strand DNA replication in eukaryotes. DNA Pol ε in yeast consists of four subunits, Pol2, Dpb2, Dpb3, and Dpb4. Pol2 is the catalytic subunit and Dpb2, Dpb3, and Dpb4 are the accessory subunits. Pol2 can be further divided into an N-terminal catalytic core (Pol2core) containing both the polymerase and exonuclease active sites and a C-terminus domain. We determined the X-ray crystal structure of Pol2core at 2.2 Å bound to DNA and with an incoming dATP. Pol ε has typical fingers, palm, thumb, exonuclease, and N-terminal domains in common with all other B-family DNA polymerases. However, we also identified a seemingly novel domain we named the P-domain that only appears to be present in Pol ε. This domain partially encircles the nascent duplex DNA as it leaves the active site and contributes to the high intrinsic processivity of Pol ε. To ask if the crystal structure of Pol2core can serve as a model for catalysis by Pol ε, we investigated how the C-terminus of Pol2 and the accessory subunits of Pol ε influence the enzymatic mechanism by which Pol ε builds new DNA efficiently and with high fidelity. Pre-steady state kinetics revealed that the exonuclease and polymerization rates were comparable between Pol2core and Pol ε. However, a global fit of the data over five nucleotide-incorporation events revealed that Pol ε is slightly more processive than Pol2 core. The largest differences were observed when measuring the time for loading the polymerase onto a 3' primer-terminus and the subsequent incorporation of one nucleotide. We found that Pol ε needed less than a second to incorporate the first nucleotide, but it took several seconds for Pol2core to incorporate similar amounts of the first nucleotide. B-family polymerases have evolved an extended β-hairpin loop that is important for switching the primer terminus between the polymerase and exonuclease active sites. The high-resolution structure of Pol2core revealed that Pol ε does not possess an extended β-hairpin loop. Here, we show that Pol ε can processively transfer a mismatched 3' primer-terminus between the polymerase and exonuclease active sites despite the absence of a β-hairpin loop. Additionally we have characterized a series of amino acid substitutions in Pol ε that lead to altered partitioning of the 3'primer-terminus between the two active sites. In a final set of experiments, we investigated the ability of Pol ε to displace the downstream double-stranded DNA while carrying out DNA synthesis. Pol ε displaced only one base pair when encountering double-stranded DNA after filling a gap or a nick. However, exonuclease deficient Pol ε carries out robust strand displacement synthesis and can reach the end of the templates tested here. Similarly, an abasic site or a ribonucleotide on the 5'-end of the downstream primer was efficiently displaced but still only by one nucleotide. However, a flap on the 5'-end of the blocking primer resembling a D-loop inhibited Pol ε before it could reach the double-stranded junction. Our results are in agreement with the possible involvement of Pol ε in short-patch base excision repair and ribonucleotide excision repair but not in D-loop extension or long-patch base excision repair.

Thesis (M.S.)--Miami University, Dept. of Chemistry and Biochemistry, 2004. / Title from first page of PDF document. Includes bibliographical references (p. 57-64).

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Natural products are secondary metabolites produced by plants and microorganisms that often possess medicinal properties and are implicated in organismal defense. Drawbacks to utilizing natural products in the pharmaceutical industry are difficulties with isolation from biological sources and low yields that can lack stereospecificity from synthetic sources. It is paramount to solve these issues and to develop novel natural products to combat the growing antimicrobial resistance crisis, which was responsible for ~5 million deaths in 2019 alone. One approach is utilizing enzymes to synthesize existing natural products to improve the yields and stereospecificity issue. This dissertation is focused on the biochemical characterization of three enzymes-ZvFMO, OxaD, and CreE-that are implicated in the detoxification of natural products used for organismal defense or participate in the biosynthesis of novel natural products. Each of these enzymes belong to the flavin-dependent monooxygenase (FMO) family, which catalyze the oxygenation of a substrate, generating an oxidized product. ZvFMO, from the insect food crop pest, Zonocerus variegatus, was determined to catalyze a highly uncoupled oxygenation reaction of the nitrogen or sulfur atom of various substrates. OxaD, from Penicillium oxalicum F30, catalyzes novel sequential oxidation reactions of the indole nitrogen of roquefortine C. CreE, from Streptomyces cremeus, also catalyzes sequential nitrogen oxidation reactions to convert L-aspartate to nitrosuccinate en route to biosynthesis of cremeomycin. For each enzyme, the steady-state kinetics have been determined using an oxygen consumption assay and the rapid-reaction kinetics were measured using anaerobic time-resolved spectroscopy. All three enzymes feature a fast flavin reduction step and a slow flavin dehydration step. The oxygenation chemistry of each enzyme was found to proceed through a highly reactive oxygenating species, the C4a-hydroperoxyflavin. Site-directed mutagenesis efforts led to the identification of key active site residues involved in flavin motion and substrate binding, revealing important information about the active site architecture for enzyme engineering applications and drug discovery efforts. / Doctor of Philosophy / Natural products are compounds that are produced by many plants, fungi, and bacteria that have potent medicinal properties and can be used to defend the organism against pests. Unfortunately, using these compounds widely in the pharmaceutical industry is difficult because it is hard to isolate the compound of interest from the organism that produces it and attempts to produce it chemically can result in low yields. Additionally, the overuse of the current natural products, which are most of the antibiotics on the market today, has led to an extreme increase in the resistance of bacteria, fungi, and parasites to the natural product-based drug. Therefore, it is essential that a method is developed to produce novel natural products at high yields to combat the antimicrobial resistance crisis. One method is by using enzymes to generate the natural products of interest. Enzymes are biological catalysts that speed up reactions by ensuring that less energy is required to transition from a reactant to a product and are highly efficient. This dissertation focuses on the characterization of three enzymes that could aid in our understanding of natural product chemistry. All three enzymes insert an oxygen atom on a nitrogen of their respective reactant. The first enzyme ZvFMO, is from an insect and its reactivity causes the insect to become resistant to the natural product-based plant defense mechanism, demonstrating that ZvFMO is a great candidate for inhibitor design. OxaD is the second enzyme and is involved in producing natural products that have antimicrobial and anticancer properties. The last enzyme, CreE, is involved in generating the natural product, cremeomycin, which possesses potent antimicrobial and anticancer properties as well. The reactions of OxaD and CreE positions these enzymes as candidates to produce novel natural products and other efforts to expand their reactivity. The rates of each reaction step have been determined in this work. Key amino acids that contribute to the reaction chemistry and the uptake of the reactant have been identified, laying a solid foundation for drug discovery efforts.

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