Much evidence now exists to show that unbalanced DNA precursor pools cause DNA replicational infidelity in vitro. However, there are relatively few data with in vivo systems. Experiments were performed therefore, to determine if unbalanced precursor pools could be induced in vivo and if so, what affect this would have on various genetic markers. The nucleoside thymidine was shown to be completely non-toxic to the rat when administered orally, negative in the dominant lethal assay and was only marginally clastogenic in the micronucleus test. Treatment of human lymphocyte chromosomes with the essential amino acid arginine, arrested cell division possibly due to a predominance of arginine-rich histones limiting the chromatin-condensation during mitosis. Thymidine administered i. p. to mice induced marked increases in the proportions of abnormal sperm and the same effect, to a lesser extent, was seen in rats. The affected germ-cell stages were the mid - to late pachytene spermatocytes. These affects were probably due to base-misincorporation occuring during unscheduled DNA synthesis. The purine nucleoside adenine caused dose-related increases in the frequency of abnormal sperm in mice. In rats, a proportion of animals given 150mg/kg adenine showed high levels of abnormal sperm whilst others were unaffected. Examination of mice in the generation revealed that the damage to the germ-cells was transmissible. The simultaneous administration of deoxycytidine with excess thymidine to mice partly inhibited the effects on sperm morphology indicating that those effects were due to precursor pool imbalance. In addition, an analytical technique was developed to measure nucleosides and bases in the testes using HPLC. The method proved to be rapid, reproducible and quantitative and showed that 1hr following i. p. injection, thymidine levels in the testes increased markedly and thereafter quickly return to control levels. Finally, experiments were initiated to investigate the mechanisms underlying the formation of morphogically abnormal sperm. Polyacrylamide gel electrophoresis was used to identify membrane proteins in sperm from both control and treated animals.

Dans les trois domaines du vivant, que constituent les bactéries, les eucaryotes et les archées, une molécule a la capacité souveraine de gouverner la vie, la mère à l’origine de tous les mécanismes biologiques, l’ADN. S’il est évident de dire que le maintien de l’intégrité des génomes est essentiel à la vie, il existe deux systèmes qui le permettent, la réplication et la réparation de l’ADN. La fidélité de ces derniers est finement influencée par la disponibilité (ratio et balance) des précurseurs nucléotidiques désoxyribonucléotides (dNTPs) et ribonucléotides (rNTPs) au cours du cycle cellulaire. Même si la concentration intracellulaire en nucléotides est largement documentée chez les eucaryotes et les bactéries, ça n’est malheureusement pas le cas chez les archées. En ce qui concerne l’étude de la maintenance génomique, un groupe d’archées a intéressé les chercheurs de par leurs capacités à survivre dans des milieux dits extrêmes. Pyrococcus abyssi est l’une d’entre elles qui depuis de nombreuses années sert de modèle biologique pour répondre aux questions de la stabilité de l’ADN à haute température. Cette étude est centrée sur cette thématique et particulièrement sur les caractéristiques fonctionnelles des ADN polymérases: PolD, PolB et le complexe p41/p46. Initialement, le contenu en nucléotides a été évalué dans des cellules en phase exponentielle de croissance par la technique de chromatographie couplée à une double détection en spectrométrie de masse (zicHILIC-MS-MS). Les résultats montrent que le contenu en rNTPs est de 20 fois supérieur à celui en dNTPs. Pour cette raison, la discrimination sélective des dNTPs par les ADN polymérases est mise à l’épreuve. Même si, des mécanismes permettent d’exclure les rNMPs durant la synthèse de l’ADN, de récentes études ont montrées que des rNMPs étaient incorporés par des ADN pols. Ainsi, le ratio en nucléotides obtenu a été utilisé pour l’analyse de son effet sur la synthèse d’ADN par les ADN Pols et les extraits cellulaires de P. abyssi. Les résultats démontrent clairement que les rNMPs sont incorporés par l’ADN polymérase PolD. Puis, les conséquences de la présence des rNMPs dans l’ADN sur la réplication ont été étudiées et ont mis en évidence que les extraits cellulaires, tout comme les ADN Pols de P. abyssi étaient capables de « passer » un rNMP présent dans l’ADN. Pour finir, une étude de l’incorporation préférentielle de chaque dNMP et rNMP a été menée démontrant que la complémentarité des bases était respectée même lors de l’incorporation de rNMPs. Enfin, la caractérisation de la petite sous-unité, DP1, de PolD a permis de montrer sa capacité à retirer des rNMPs grâce à son activité de relecture, suggérant un premier rempart à la présence de rNMPs dans l’ADN. Pour conclure, ces résultats montrent que la présence de rNMPs dans l’ADN est un phénomène conservé dans les trois domaines du vivant. / In the three domains of life that include Bacteria, Eukarya and Archaea, one molecule has the sovereign ability to govern life, and not the least one, the mother of all biological mechanisms, DNA. Maintaining the integrity of genomes is obviously essential for life, and faithful DNA replication and repair are the guarantees. The fidelity of these two processes may vary depending on the availability and levels (balance and ratio) of deoxyribonucleotides (dNTPs) and ribonucleotides (rNTPs) during the cell-cycle. Even if intracellular concentration of nucleotides is largely documented in Eukarya and Bacteria, it remains limited in Archaea. From many years one group of Archaea is of great interest for studying genomic maintenance, because of its ability to survive in extremes environments. Pyrococcus abyssi is one of them that is used as biological model for deciphering the stability of DNA at elevated temperature in LM2E. The present work focuses on genomic integrity and particularly on the functional characterization of the three DNA polymerases: PolD, PolB and the p41/p46 complex. Initially, the nucleotide pool has been evaluated in exponentially growing cells using the highly sensitive method that combined chromatography and mass spectrometry (zicHILIC-MS-MS). The results show that rNTPs content is 20-fold higher than dNTPs. For that reason, fidelities of DNA polymerases are challenged to select the correct dNTP over the most abundant rNTP during DNA synthesis. Despite the fact that some mechanisms allow the exclusion of rNTPs from entry to the Pol active site, recent findings indicate that ribonucleotides are incorporated by different DNA Pols with surprisingly high frequency. In this work, the obtained intracellular balance and ratio of rNTPs and dNTP have been used to analyze their effect on DNA synthesis by P. abyssi DNA Pols and cell-free extracts. Our results clearly demonstrate that rNTP incorporation is detectable with distinct efficiencies among DNA pols. Secondly, the consequences of the presence of rNMPs in a DNA template on DNA polymerisation has been examined and highlights that cell-free extracts are able to bypass a single rNMP as well as replicative DNA polymerases. To strengthen that study, single nucleotide incorporation opposite rNMP or dNMP has been carried out and the results demonstrate that replicative Pyrococcus abyssi DNA Pols can basepair the complementary rNTPs opposite dNMPs, and vice-versa, the complementary dNTPs opposite rNMPs.Furthermore, the preliminary results obtained about the nucleolysis activities of the PolD small subunit, DP1, show that the DNA polymerase D is able to remove rNMPs from a DNA strand, suggesting a first level of protection against ribonucleotide contamination of DNA. Definitely, these data indicate that the presence of transient embedded rNMPs in genomic DNA represents a universally conserved phenomenon across Archaea, Bacteria and Eukarya.

La stabilité du génome est compromise par les déséquilibres du pool de dNTPs qui affectent la vitesse de progression des fourches de réplication. Par exemple, la déficience en cytidine désaminase (CDA) conduit à un excès de dCTP qui induit un ralentissement des fourches de réplication. Les résultats obtenus au cours de ma thèse ont permis de mettre en évidence un nouveau mécanisme par lequel un déséquilibre du pool de nucléotides compromet la complétion de la réplication et la ségrégation correcte des chromosomes. En utilisant des techniques de peignage moléculaire, de microscopie électronique et d’imagerie cellulaire permettant de quantifier le niveau basal de PAR, nous avons montré que la réplication incomplète de l’ADN lorsque la CDA est absente est due à l’inhibition partielle de PARP-1, et n’est pas liée au ralentissement de la vitesse de progression des fourches de réplication. En effet, l’accumulation intracellulaire de dCTP inhibe l’activité de PARP-1 ce qui réduit l’activation de Chk1 et l’efficacité des points de contrôle situés en aval, favorisant ainsi l’accumulation de séquences d’ADN non répliquées en mitose. Celles-ci conduisent alors à la formation de ponts anaphasiques ultrafins (UFBs) entre les chromatides sœurs au niveau de sites difficiles à répliquer tels que les centromères et les sites fragiles. Ces résultats ont des implications directes dans le syndrome de Bloom (BS), une maladie génétique rare combinant prédisposition aux cancers et instabilité génétique. Ce syndrome est la conséquence de la mutation du gène BLM, codant pour une hélicase RecQ du même nom. La déficience en BLM conduit à une chute de l’expression de la CDA résultant en une augmentation des UFBs entièrement due à l’inhibition de PARP-1 par la dCTP, indépendamment de BLM. Ces travaux décrivent ainsi une conséquence pathologique encore inconnue du déséquilibre du pool de nucléotides et révèlent un rôle inattendu de PARP-1 dans la surveillance des séquences d’ADN non répliquées prévenant leur accumulation en mitose et les défauts de ségrégation des chromosomes associés. / Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that in CDA-deficient cells DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. Indeed, the intracellular accumulation of dCTP inhibits PARP-1 activity compromising the activation of Chk1 and the downstream checkpoints efficiency, allowing the subsequent accumulation of unreplicated DNA in mitosis. This unreplicated DNA leads to the formation of ultrafine anaphase bridges (UFBs) between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of UFBs due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our results describe previously unknown pathological consequences of the distortion of dNTP pools and reveal an unexpected role for PARP-1 in preventing unreplicated DNA accumulation in mitosis and in preventing chromosome segregation defects.

Diet and oral supplements comprise two distinct sources of antioxidants known to prevent oxidative stress. Beneficial effects from antioxidants have been seen for patients at risk for type 2 diabetes. The aim of this thesis was to evaluate the positive effects of antioxidants against oxidative stress and DNA damage in type 2 diabetes subjects. We also used antioxidants as tools to determine the mechanisms behind genotoxicity induced by mutagenic pro-oxidative agents in mouse lymphoma cells. Several techniques were used to measure oxidative stress and DNA damage, but the main technique used was alkaline comet assay. The results showed that the fruit and vegetable intake was inversely related to oxidative stress in type 2 diabetes subjects. However, oral supplementary intake of 20 antioxidants did not decrease oxidative stress biomarkers. In studies on mouse lymphoma cells, using the alkaline comet assay, DNA damage was induced by catechol and o-phenylenediamine (OPD), while 4-nitro-o-phenylenediamine (4-NOPD) induced only oxidative damage, showing different mechanisms of action behind the mutagenicity of the compounds. Also, oxidative stress was induced by catechol and 4-NOPD, whereas imbalances in the nucleotide pool were seen after exposure to OPD or 4-NOPD. Addition of antioxidants together with these pro-oxidants showed that β-carotene was able to reduce DNA damage at low concentrations of catechol, but increased DNA damage at high concentration. In comparison, addition of α-tocopherol slightly decreased catechol-induced DNA damage at all concentrations of catechol. However, no effect of α-tocopherol was seen on OPD-or 4-NOPD-induced DNA damage. In conclusion, antioxidants from fruits and vegetables, but not from oral supplements, reduced oxidative stress in type 2 diabetes patients, suggesting fruits and vegetables being a healthier source for antioxidant-intake, as compared to oral supplements. Different mechanisms of action for mutagenic pro-oxidants were shown in mouse lymphoma cells, introducing the nucleotide pool as an interesting target for oxidative stress. Reduction of catechol-induced DNA damage by β-carotene or α-tocopherol was shown, with a pro-oxidative action of β-carotene at high concentration of catechol, Interestingly, α-tocopherol was not able to decrease OPD- or 4-NOPD-induced DNA damage, supporting different mechanisms of action behind the genotoxicity from the three pro-oxidants.