The human mitochondrial polymerase (pol γ) is a nuclearly-encoded polymerase that is solely responsible for the faithful replication and repair of the mitochondrial genome. The Y955C mutation in pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson’s disease. It is believed that the position of this Y955 residue on the catalytic helix in the polymerase makes it responsible for stabilizing the incoming nucleotide. I have investigated the kinetic effect of the Y955C mutation. Mutation of the tyrosine to a cysteine resulted in a decreased maximum rate of polymerization and increased the dissociation constant for incoming nucleotide. In turn, this decreased catalytic efficiency by 30 to 100-fold. In addition, the polymerase did not incorporate all bases with the same efficiency, it was most efficient when incorporating dGTP opposite a dC, but showed less efficient catalysis when faced with an A:T or T:A base-pair. The polymerase also showed reduced discrimination against misincorporation events. However, when presented with an oxidatively-damaged base, 8-oxo-deoxyguanosine, the polymerase chose to incorporate the base in the correct conformation opposite a dC, discriminating against the mutagenic incorporation of 8-oxo-dGTP opposite a dA. The results presented in this thesis suggest that the severe clinical symptoms of patients with this mutation are at least due in part to the reduced efficiency and discrimination of this polymerase γ mutation. / text

The human mitochondrial DNA (mtDNA) genome must be faithfully maintained by the mitochondrial DNA replication machinery. Deficiencies in mtDNA maintenance result in the accumulation of mutations and deletions, which have been associated with a number of neuromuscular degenerative disorders including, mtDNA depletion syndrome, Alpers syndrome, progressive external opthalmoplegia (PEO), and sensory ataxic neuropathy, dysarthria, and opthalmoparesis (SANDO). The mtDNA replication machinery is comprised of a nuclearly-encoded DNA polymerase gamma (Pol γ), single-stranded DNA binding protein (mtSSB), and a hexameric mtDNA helicase. In this work, we employed quantitative pre-steady state kinetic techniques to establish the mechanisms responsible for the replication of the human mitochondrial DNA by Pol γ and explored the effects of point mutations that are observed in heritable diseases. With our biochemical characterization of mutants of Pol γ, we have shown unique characteristics that would lead to profound physiological consequences over time. Additionally, we have made significant progress towards reconstitution of the mitochondrial DNA replisome by monitoring DNA polymerization that is dependent on helicase unwinding of double stranded DNA. Overall, this work provides a better understanding of the mechanism of mtDNA replication and has important implications toward understanding the role of mitochondrial DNA replication in mitochondrial disease, ageing and cancer. In addition to the work on the mtDNA replisome, we have applied pre-steady state kinetic techniques to better understand the mechanism of RNA-dependent DNA polymerization by HIV reverse transcriptase (HIV-RT). This enzyme is responsible for the replication of the viral genome in HIV and is a common target for anti-HIV drugs. We have characterized the role of enzyme conformational changes in the kinetics of incorporation of correct nucleotide and the Nucleotide Reverse Transcriptase Inhibitor (NRTI) AZT by wild-type enzyme, as well as a mutant with clinical resistance to AZT. This work provides a better understanding of the complete mechanism of RNA-dependent DNA polymerization, the changes in the mechanism in the presence of inhibitor and the development of resistance to this nucleoside analog; and thereby this work contributes to the long-term goal of designing more effective drugs that can possibly deter resistance and be used successfully for treatment of HIV. / text

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Nitroreductase from Enterobacter cloacae (NR) is a member of a large family of homologues represented in all branches of the tree of life. However the physiological roles of many of these enzymes remain unknown. NR has distinguished itself on the basis the diverse sizes and chemical types of substrates it is able to reduce (Koder et al 1998). This might be an evolved characteristic suiting NR for a role in metabolism of diverse occasional toxins. While there are numerous studies of determinants of substrate specificity, we know less about mechanisms by which enzymes can be inclusive. Therefore, we present a synthesis of NR's dynamics, stability, ligand binding repertoire and kinetic mechanism. We find that NR reduces para-nitrobenzoic acid (p-NBA) via a simple mechanism limited by the chemical step in which the nitro group is reduced (Pitsawong et al 2014). Thus, for this substrate, NR's mechanism dispenses with gating steps that in other enzymes can enforce substrate specificity. Our data demonstrate that substrate reduction is accomplished by rate-contributing hydride transfer from the flavin cofactor coupled to proton transfer from solvent, but do not identify specific amino acids with a role. This is consistent with our crystal structures, which reveal a spacious solvent-exposed active site bounded by a helix that moves to accommodate binding of substrate analogs (Haynes et al 2002). Because it is able to reduce TNT (trinitrotoluene), herbicides and pesticides, NR has important potential utility in bioremediation.

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.

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