Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in cells. In an in vitro replication system involving semiconservative replication from an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human cell extracts in reaction mixtures containing "biologically biased" dNTP pools estimated from HeLa cell nuclei is not significantly different from that seen when replication is done with equimolar dNTP concentrations. Significant reduction of dGTP pool while keeping other dNTPs at "biologically biased" dNTP concentrations during replication reaction also did not increase mutation frequency. In contrast, in vitro replication with dNTP concentrations calculated from normal diploid fibroblast cells, which are three- to four-fold lower in concentrations, showed a marked reduction of the observed mutation frequency, showing the importance of overall dNTP levels during replication on mutation frequency in vitro. When whole-cell dNTP concentrations in HeLa cells were measured during S-phase, dNTP levels underwent a transient decrease in the middle of S-phase. Average HeLa cells' dNTP levels were also found to correlate with average DNA accumulation rates during S-phase, although no detailed relationship can yet be deducted from the available data. No significant changes in the ratio of the four dNTP concentrations were found during S-phase. Mutation rates of green fluorescent protein (GFP) inserted in either middle or late-replicating region of a chromosome in HeLa cells also correspond to average DNA accumulation rates and dNTP levels during middle and late S-phase. The late-replicating GFP-HeLa cells have a higher mutation rate than the middle-replicating GFP-HeLa cells, as the average DNA accumulation rates and dNTP pool levels were also lower in the middle compared to late S-phase. Taken together, these observations indicate that dNTP levels could play a role in determining the S-phase DNA replication rate and also the replication fidelity in mammalian cells. / Graduation date: 2002

No description available.

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

Chez les eucaryotes supérieurs, la réplication de l'ADN est initiée à partir de plusieurs milliers d'origines. Mais la régulation spatio-temporelle de leur activation reste mal caractérisée. Une voie de contrôle (checkpoint) de la phase S est activée lorsque les fourches de réplication sont bloquées, inhibant ainsi l'activation d'origines tardives. L'objectif de ma thèse consistait à étudier deux facteurs essentiels dans le programme spatio-temporel de la réplication, dans le système du xénope : la protéine « checkpoint » Chk1, qui est un facteur inhibiteur de l'activation des origines, et les désoxyribonucléotides (dNTPs), précurseurs de la synthèse de l'ADN. Chez le xénope, après douze divisions embryonnaires, a lieu la transition mid-blastuléenne (MBT). A cette étape, une augmentation du ratio nucléo-cytosolique va entrainer la titration des facteurs de réplication, ce qui active le point de contrôle et ralentit la phase S. Il est possible de mimer in vitro les phases S rapides des embryons pendant le développement précoce en augmentant la concentration en noyaux dans l'extrait d'œufs.Nous avons pu voir par l'inhibition, la déplétion ou la surexpression de Chk1 que cette protéine régulait l'activation des origines lors d'un stress, mais également dans une phase S non perturbée, grâce à la technique du peignage moléculaire. Ce résultat montre que le niveau de Chk1 doit être finement régulé pour permettre une réplication correcte dans une phase S non perturbée, chez les eucaryotes supérieurs. Nous avons ensuite cherché à savoir si la concentration en dNTPs pouvait être limitante pendant le développement et comment elle modulait le programme de réplication. Nous avons comparé l'effet de l'ajout de dNTPs sur la réplication en mimant plusieurs stades précoces du développement pré-MBT. La variation de la concentration en dNTPs agit sur la réplication en augmentant à la fois l'activation des origines et, en fonction de la concentration en noyaux, aussi la vitesse des fourches. Cet effet est indépendant du checkpoint de la réplication dans ce système et d'autres études sont nécessaires pour comprendre les mécanismes moléculaires. / DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal regulation program which is not well characterized. The S phase checkpoint is activated when replication forks are blocked which inhibits the firing of late origins. The aim of my thesis consisted to study two essentials factors in spatio-temporal replication program in Xenopus system: the checkpoint protein Chk1, inhibitor of origin activation, and the deoxyribonucleotides (dNTPs), DNA synthesis precursors. In Xenopus, the mid-blastula transition (MBT) occurs after twelve embryonic divisions. An increase of the nucleo-cytosolic ratio induces a titration of replication factors, that activates the checkpoint and slows down the S phase. It is possible to mimic in vitro the rapid S phases of early Xenopus development stages by increasing the nuclei concentration. By DNA combing combined with Chk1 inhibition, depletion and overexpression experiments, we show that Chk1 controls origins activation in perturbed but also unperturbed S phase. My results show that Chk1 levels needs to be tightly regulated in order to properly control the replication program during normal S phase in higher eukaryotes. In order to determine whether the concentration of dNTPs could be another limiting replication factor, we compared the effect of dNTPs addition on replication by mimicking in vitro several early stages of pre-MBT development. Addition of dNTPs affects DNA replication, by increasing origin activation and, dependent on nuclei concentration, also the fork speed. This effect is independent of the S phase checkpoint and further studies are needed in order to understand the molecular mechanisms behind.

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