The faithful replication of DNA depends on the appropriate balance of DNA precursors. From studies conducted in bacteriophage T4, models for deoxyribonucleotide biosynthesis producing pools appropriate for DNA replication have made it possible to understand more complex systems. A portion of that body of evidence supports the concept that deoxyribonucleotide biosynthesis for bacteriophage T4 is carried out by an association of enzymes and other cellular components in a complex called the dNTP synthetase complex. This dissertation explores potential direct protein-protein interactions within this complex for the preparation of pyrimidine deoxyribonucleotides. Direct associations for enzymes involved in pyrimidine deoxyribonucleotide biosynthesis were examined by affinity chromatography. It was determined that there was a significant direct relationship between T4 thymidylate synthase and T4 dCMP deaminase, between T4 dCTPase/dUTPase and T4 dCMP deaminase as well. The interaction between thymidylate synthase and dCMP deaminase was significantly influenced by the presence of dCTP, a positive effector of dCMP deaminase. Furthermore, protein associations changed the kinetic character of pyrimidine deoxyribonucleotide production. T4 dCTPase/dUTPase, a member of the dNTP synthetase complex, significantly alters the kinetic nature of thymidylate synthase by working with thymidylate synthase in a reciprocal relationship. T4 single-stranded DNA binding protein, a member of the replication complex, alters the activity of thymidylate synthase as well. Attempts to isolate a kinetically coupled complex from two or more constituent proteins of the dNTP synthetase complex were frustrated by protein degradation to fragments under 10 kDa in size. Pyrimidine deoxyribonucleotide synthesis is located between the significant energy investment of ribonucleotide reductase and phosphate attachments by kinases to prepare the deoxyribonucleotide molecules for DNA replication. In bacteriophage T4, intermediate reactions are driven by mass action but are modulated by subtleties including direct protein associations and the presence of small molecules that influence enzyme function. Through these and potentially similar controls, pools of deoxyribonucleotides are prepared and delivered in a timely, balanced manner to the DNA replication apparatus. / Graduation date: 2002

It is well known that the mitochondrial genome has a much higher spontaneous mutation rate than the nuclear genome. mtDNA mutations have been identified in association with many diseases and aging. mtDNA replication continues throughout the cell cycle, even in post-mitotic cells. Therefore, a constant supply of nucleotides is required for replication and maintenance of the mitochondrial genome. However, it is not clear how dNTPs arise within mitochondria nor how mitochondrial dNTP pools are regulated. Recent evidence suggests that abnormal mitochondrial nucleoside and nucleotide metabolism is associated with several human diseases. Clearly, to uncover the pathogenesis of these diseases and the mechanisms of mitochondrial mutagenesis, information is needed regarding dNTP biosynthesis and maintenance within mitochondria, and biochemical consequences of disordered mitochondrial dNTP metabolism. The studies described in this thesis provide important insight into these questions. First, we found that a distinctive form of ribonucleotide reductase is associated with mammalian liver mitochondria, indicating the presence of de novo pathway for dNTP synthesis within mitochondria. Second, we found that long term thymidine treatment could induce mtDNA deletions and the mitochondrial dNTP pool changes resulting from thymidine treatment could account for the spectrum of mtDNA point mutations found in Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients. These results support the proposed pathogenesis of this disease. Third, we found that normal intramitochondrial dNTP pools in rat tissues are highly asymmetric, and in vitro fidelity studies show that these imbalanced pools can stimulate base substitution and frameshift mutations, with a substitution pattern that correlates with mitochondrial substitution mutations in vivo. These findings suggest that normal intramitochondrial dNTP pool asymmetries could contribute to mitochondrial mutagenesis and mitochondrial diseases. Last, Amish lethal microcephaly (MCPHA) has been proposed to be caused by insufficient transport of dNTPs into mitochondria resulting from a loss-of-function mutation in the gene encoding a mitochondrial deoxynucleotide carrier (DNC). We found that there are no significant changes of intramitochondrial dNTP levels in both a MCPHA patient's lymphoblasts with a missense point mutation in Dnc gene and the homozygous mutant cells extracted from Dnc gene knockout mouse embryos. These results do not support the proposed pathogenesis of this disease and indicate that the DNC protein does not play a crucial role in the maintenance of intramitochondrial dNTP pools. / Graduation date: 2005

The mechanism for the hydrolysis of 2′-deoxyribonucleosides is examined using computational chemistry techniques. Initially, a model capable of accurately predicting the mechanism and activation barrier for the uncatalyzed hydrolysis of 2′-deoxyuridine is designed. It is found that the smallest model includes both explicit and implicit solvation during the optimization step. Next, this hybrid solvation model is applied to four natural nucleosides, namely 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine and thymidine. The hybrid model correctly predicts the trend in activation Gibbs energies for the pyrimidines and purines, separately. Finally, the concepts developed during the generation of the uncatalyzed hydrolysis model are applied to the mechanism of action of a glycosylase enzyme, namely human uracil DNA glycosylase. A hybrid ONIOM approach is utilized to study the experimentally proposed two-step mechanism. Results regarding the protonation state of His148 are inconclusive, and future directions are proposed. / xiii, [131] leaves : ill. (some col.) ; 29 cm

With respect to a multienzyme complex of deoxyribonucleoside triphosphate (dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory has demonstrated the existence of a multienzyme complex in T4-infected E. coli, named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP synthesis to DNA replication and organization of the complex has not been well established. This study had two objectives. The first objective was to test the specific hypothesis that gp32, the single-stranded DNA binding protein encoded by gene 32, plays a role in recruiting enzymes of dNTP synthesis to the replisome and in organizing the dNTP synthetase complex. By use of two newly created gene 32 mutants along with several experimental approaches, DNA-cellulose chromatography, coimmunoprecipitation, and glutathione-S-transferase pull downs, interactions of gp32 with thymidylate synthase (gptd), ribonucleotide reductase (gpnrdA/B), and E. coli NDP kinase have been identified. These results support the hypothesis that gp32 helps to recruit enzymes of dNTP synthesis to DNA replication sites. As the second objective, I investigated contributions of two host proteins, E. coli nueleoside diphosphate kinase (NDP kinase) and adenylate kinase (Adk), to the organization of the complex. As an important step to understand roles of E. coli NDP kinase in the complex, I identified direct interactions of E. coli NDP kinase with gpnrdA/B, dCMP hydroxymethylase (gp42), and dihydrofolate reductase (gpfrd) by means of coimmunoprecipitation and glutathione-S-transferase pull-down experiments. Interestingly, these interactions were influenced by the presence of substrate nucleotides or an analog for E. coli NDP kinase, suggesting that metabolite flux may affect the preference of E. coli NDP kinase binding to enzymes in the complex in vivo. Meanwhile, Adk involvement in DNA precursor synthesis has been suggested, particularly in phage T4-infected E. coli, from observations of increased thermostability of temperature-sensitive Adk in situ. The involvement of E. coil Adk in the complex was demonstrated by identifying some proteins of the T4 dNTP synthetase complexgp42, dNMP kinase (gpl), gpfrd, and E. coli NDP kinasedirectly interacting with Adk, implying that E. coil Adk would be properly located in the complex to efficiently carry out the conversion of dNDPs to dNTPs. This implication was supported by measurements of T4 DNA synthesis. Taken together, this research strongly supports the idea of connection of dNTP synthesis to DNA replication and allows us to take a step toward understanding the organization of the complex at DNA replication sites. / Graduation date: 2005