Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The first study characterized the expression of MAS receptor and key enzymes for Ang-(1-7) production, such as, ACE2, NEP and PEP during follicular development. Furthermore, the regulation of local Ang1-7 system was evaluated after the intrafollicular injection of fulvestrant (an estradiolreceptor inhibitor) in the dominant follicle. Cows were ovariectomized when the size between the largest (F1) and the second largest follicle (F2) was not statistically different (Day 2), slightly different (Day 3), or markedly different (Day 4). The mRNA abundance of genes encoding MAS receptor, ACE2, NEP and PEP was evaluated in the follicular cells from F1 and F2. The mRNA expression of MAS receptor was upregulated in the granulosa cells of F2 after the establishment of follicular deviation (Day 4), while PEP mRNA increased during (Day 3) and after (Day 4) the deviation process. However, the mRNA expression of ACE2 was upregulated in the granulosa cells of F1 during and after the deviation process. The mRNA expression of NEP was not regulated in F1 and F2. The MAS receptor was immunolocated in the granulosa and theca cells of F1 and F2 during follicular deviation. Moreover, MAS receptor gene expression increased when the F1 was treated with the estrogen receptor-antagonist in vivo. In conclusion, the expression profile of MAS receptor, ACE2, NEP and PEP in dominant and subordinate follicles indicated that Ang-(1-7) play a role in the regulation of the follicular dominance in cattle. A second study was performed to investigate the expression of genes that control homologous recombination (HR; 53BP1, ATM, RAD50, RAD51, RAD52, BRCA1, BRCA2 and NBS1), and non-homologous end-joining (NHEJ; KU70, KU80 and DNAPK), DNArepair pathways in bovine embryos with high, intermediate or low developmental competence. We also evaluated whether bovine embryos can respond to DNA double-stranded breaks (DSBs) induced by ultraviolet (UV) irradiation by regulating the expression of genes involved in the HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro fertilization and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation (EGA). All studied genes were expressed before, during and after the EGA period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before EGA in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-fertilization in bovine embryos with DSBs induced by UV irradiation. In conclusion, important genes controlling HR and NHEJ repair pathways are expressed in bovine embryos before, during or after EGA. Lower developmental competence seems to be associated with a higher mRNA expression of 53BP1 and RAD52. Bovine embryos can response to UV-induced DSBs after the EGA but HR and NHEJ repair pathways seem to be particularly regulated at the blastocyst stage. / O primeiro estudo caracterizou a expressão do receptor MAS e de enzimas responsáveis pela produção de Ang-(1-7), tais como, enzima conversora de angiotensina 2 (ACE2), endopeptidase neutra (NEP) e prolil endopeptidase (PEP) durante o desenvolvimento folicular. Além disso, a regulação local do sistema Ang-(1-7) foi avaliada após a injeção intrafolicular de fulvestrant (inibidor do receptor de estradiol) no folículo dominante. As vacas foram ovariectomizadas quando o tamanho entre o maior (F1) e o segundo maior folículo (F2) não era estatisticamente diferente (D2), ligeiramente (D3) ou marcadamente diferente (D4). A expressão de RNAm do receptor MAS, ACE2, NEP e PEP foi avaliada nas células foliculares do F1 e F2. O receptor MAS foi mais expresso nas células da granulosa do F2 após o estabelecimento da divergência folicular (D4), enquanto a expressão de PEP aumentou durante (D3) e após (D4) o processo de divergência. Entretanto, a expressão de ACE2 foi maior nas células da granulosa do F1 durante e após a divergência. A expressão de PEP não foi regulada no F1 e F2. O receptor MAS foi imunolocalizado nas células da teca e granulosa do F1 e F2 durante a divergência folicular. A expressão de RNAm do receptor MAS aumentou quando o F1 foi tratado com fulvestrant in vivo. Em conclusão, o perfil de expressão do receptor MAS, ACE2, NEP e PEP nos folículos dominante e subordinado indicam que a Ang-(1-7) apresenta uma função na regulação da dominância folicular em bovinos. Em um segundo estudo investigamos a expressão de genes que controlam o reparo do DNA através das vias de recombinação homóloga (HR; 53BP1, ATM, RAD50, RAD51, RAD52, BRCA1, BRCA2, NBS1) e união terminal não homóloga (NHEJ; KU70, KU80, DNAPK) em embriões bovinos com alta, média ou baixa competência de desenvolvimento. Foi também avaliado se embriões bovinos podem responder a quebra na fita dupla de DNA (DSBs), induzida por irradiação UV, através da regulação de genes envolvidos nas vias de reparo HR e NHEJ. Embriões com alta, média ou baixa competência de desenvolvimento foram selecionados pelo tempo de clivagem após a fertilização in vitro e foram removidos do cultivo antes (36 h), durante (72 h) ou após (96 h) o momento esperado para a ativação do genoma embrionário (AGE). Todos os genes foram expressos antes, durante e após a AGE independentemente da competência de desenvolvimento dos embriões. A expressão de 53BP1 e RAD52 foi maior antes da AGE em embriões com baixa competência de desenvolvimento. A expressão de 53BP1, RAD51 e KU70 foi mais baixa as 72 h e maior as 168 h pós fertilização em embriões com DSBs induzida por irradiação UV. Em conclusão, genes importantes para o controle das vias de reparo HR e NHEJ são expressos em embriões bovinos independentemente do tempo de cultivo ou da competência de desenvolvimento. A menor competência de desenvolvimento embrionário parece estar associada com maior expressão de 53BP1 e RAD52. Os embriões bovinos respondem a DSBs após a AGE mas as vias HR e NHEJ são reguladas principalmente no estágio de blastocisto.

Les télomères sont des séquences d’ADN, généralement répétées en tandem, localisées à l’extrémité des chromosomes linéaires. Une des fonctions principales des télomères est de différencier l’extrémité des chromosomes des cassures double-brin, et ainsi de prévenir l’activation des voies de réparation de l’ADN. Chez les mammifères, cette fonction est plus spécifiquement assurée par le complexe shelterin. Il s’agit d’un complexe hétérogène composé de six protéines distinctes : TRF1, TRF2, POT1, RAP1, TPP1 et TIN2, qui interagit spécifiquement avec l’ADN télomérique. Au sein de ce complexe, les protéines RAP1 et TRF2 coopèrent afin d’empêcher l’extrémité des chromosomes d’être perçue comme un dommage de l’ADN, ce qui autrement aboutirait à des fusions inter-chromosomiques suite au processus de réparation. La protéine TRF2 se lie directement à la molécule d’ADN dans laquelle elle s’enroule de façon spécifique. Cette propriété est primordiale pour générer une structure d’ADN en forme de boucle, appelée t-loop, et dont le bon fonctionnement des télomères dépend. Les travaux effectués au cours de cette thèse ont mis en évidence deux scenarii indépendants dans lesquels la protéine RAP1 assure un rôle critique dans la stabilité des télomères. Premièrement, RAP1 peut prévenir les fusions inter-chromosomiques dans des cellules exprimant une forme altérée de TRF2 incapable de former des t-loops. Deuxièmement, l’inhibition de RAP1 dans des cellules en sénescence réplicative conduit à l’activation des voies de réparation de l’ADN et à la formation de fusions inter-chromosomiques. Ces observations font écho à des résultats précédents obtenus dans des cellules HeLa traitées avec l’inhibiteur de la télomérase BIBR1532, et dont l’expression de la protéine RAP1 était abolie par shRNA. De plus, j’ai montré que les fusions interchromosomiques engendrées par la perte de RAP1 sont dépendantes de la ligase IV, qui est un acteur principal de la voie de réparation de l’ADN par recombinaison non-homologue (NHEJ). Dans l’ensemble, ces travaux démontrent l’importance de la protéine RAP1 dans la stabilité des télomères lorsque la protéine TRF2 est non fonctionnelle, mais aussi dans des situations physiologiques telles que la sénescence réplicative. / In mammals, the shelterin complex is the guardian of telomere stability. It operates through a set of six proteins (TRF1, TRF2, POT1, RAP1, TPP1 and TIN2) that binds telomeric DNA and protects it from being recognized as DNA double-strand breaks and therefore control DNA repair and DNA damage response pathways. Among them, RAP1 and TRF2 cooperate and together protect chromosome extremities from end-to-end fusions. TRF2 is seen as a major factor to control telomere DNA topology by wrapping DNA around itself in a right handed manner. This property of TRF2 is required to promote the formation of t-loops, special DNA structures at telomeres that are considered as protective barriers to DNA damage response and fusion. Here we demonstrate two independent situations where RAP1 dysfunction is critical for telomere protection. First, in cells expressing a wrapping-deficient TRF2 allele that cannot form t-loops, RAP1 appears as a backup anti-fusion mechanism. Second, RAP1 downregulation in replicative senescent cells leads to telomere fusions and DNA damage response activation. This is consistent with similar observations in HeLa cells treated with the telomerase inhibitor BIBR1532, and in which RAP1 expression was abolished by an inducible shRNA system. In addition, we show that fusions triggered by RAP1 loss are dependent upon ligase IV, which is a key player of the classical non-homologous end-joining (c-NHEJ) repair pathway. Altogether, these results indicate that RAP1 takes over telomere protection when TRF2 cannot properly function or in the normal physiological situation, such as replicative senescence.

La voie de réparation NHEJ (Non-Homologous End-Joining) est une voie majeure de réparation des cassures double-brin chez l’homme. Les protéines de cette voie interagissent et forment des complexes dynamiques dont les mécanismes moléculaires sont encore largement méconnus. Nous avons dans un premier temps mis au point des protocoles de production à l’échelle de plusieurs milligrammes des protéines cœur de la voie NHEJ en cellules d’insecte à l’aide du système MultiBac. Nous avons ainsi purifié les complexes Ku70/Ku80 et Ligase4/XRCC4 et les protéines Cernunnos et Artemis à homogénéité. Des essais de cristallisation, des études par SAXS et des analyses par microscopie électronique ont été réalisés sur différents complexes formés par ces protéines cœur du NHEJ. Nous avons également caractérisé par chromatographie d’exclusion de taille et calorimétrie, les interactions effectuées entre les protéines de la voie NHEJ. L’ensemble de ces travaux a permis d’établir des bases biochimiques solides en vue des études structurales et fonctionnelles de la voie NHEJ chez l’homme. / Human DNA repair pathway NHEJ (Non-Homologous End-Joining) is a major pathway of double-strand breaks repair. The proteins involved in this pathway interact and form dynamic complexes whose molecular mechanisms are largely unknown. Firstly, we established protocols to be able to purify milligrams of those NHEJ pathway core proteins using MultiBac insect cells system. We then purified Ku70/Ku80 and Ligase4/XRCC4 complexes, Artemis and Cernunnos to homogeneity. Crystallogenesis assays, SAXS experiments and Transmission Electronic Microscopy experiments have been performed on several complexes formed by these core NHEJ proteins. We also characterized the interactions between these proteins by Size Exclusion Chromatography and Isothermal Calorimetry. These experiments have led to biochemical results sufficient to establish a solid basis to initiate the structural and functional study of the Human NHEJ Pathway.

Les cassures double brin de l’ADN (CDB) sont des lésions qui peuvent conduire à des réarrangements génétiques. Deux voies sont impliquées dans la réparation de ces dommages: la recombinaison homologue (HR) et la ligature d’extrémités nonhomologues (NHEJ).Au laboratoire un substrat intrachromosomique permettant de mesurer l’efficacité et la fidélité du NHEJ à été mis en place (Guirouilh-Barbat 2004). Cette approche a permis de démontrer l’existence d’une voie alternative à KU qui utilise des microhomologies présentes de part et d’autre de la cassure - le NHEJ alternatif (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). Les travaux de ma thèse consistent à caractériser les principaux acteurs de cette voie. En absence de KU, cette voie alternative du NHEJ, s'initierait tout d’abord parla résection d'extrémités d’ADN non protégées. Nous avons montré que l’activité nucléasique de MRE11 est nécessaire à ce mécanisme. La surexpression de MRE11 conduit à une stimulation du NHEJ, contrairement à l’extinction de la protéine par siRNA, résultant en une baisse de son efficacité de deux fois. Nos résultats montrent également que les protéines RAD50 et CtIP agissent dans la même voie que MRE11. De plus, dans les cellules déficientes pour XRCC4, la MIRIN – un inhibiteur du complexe MRN - conduit à une chute de l'efficacité de la réparation, démontrant le rôle de MRE11 dans la voie alternative du NHEJ. Nous avons aussi montré que MRE11 peut agir de manière dépendante et indépendante de la kinase ATM (Rass et Grabarz, Nat Struct Mol Biol 2009). L'initiation de la résection de la cassure doit être ensuite poursuivie par une dégradation plus importante de l'ADN qui est assuré par les protéines Exo1 et Sgs1/Dna2 chez la levure. Chez les mammifères, des études in vitro suggèrent un modèle similaire à deux étapes. Nous avons choisi de nous intéresser au rôle de la protéine BLM, qui est l’un des homologues humains de la RecQ hélicase Sgs1, dans la résection. Nos expériences montrent que l’absence de BLM diminue l’efficacité du NHEJ. De plus, l’extinction de BLM conduit à une augmentation d’évènements infidèles lors de la réparation par NHEJ et l’apparition d’évènements de résection de grande taille (>200nt). Ceci suggère que BLM protège contre de longues résections lors de la mise en place du NHEJ alternatif. De manière cohérente, BLM est impliquée dans la protection contre la résection dépendante de CtIP lors des étapes précoces de la recombinaison homologue. En conclusion, nos résultats montrent un rôle prédominant de BLM dans la protection contre un excès de résection médiée par CtIP. BLM interagit avec 53BP1 aux sites de dommages de manière dépendante d’ATM afin de réguler le processus de résection, en contrecarrant l’action de BRCA1. Ceci souligne à nouveau le rôle essentiel de BLM dans la protection contre la résection et la favorisation de la conversion génique sans crossing-over, ce qui est primordial pour le maintien de la stabilité du génome. / DNA double strand breaks (DSBs) are highly cytotoxic lesions, which can lead to genetic rearrangements. Two pathways are responsible for repairing these lesions : homologous recombination (HR) and non homologous end joining (NHEJ). In our laboratory, an intrachromosomal substrate has been established in order to measure the efficiency and the fidelity of NHEJ in living cells (Guirouilh-Barbat 2004). This approach led us to identify a KU-independent alternative pathway, which uses microhomologies in the proximity of the junction to accomplish repair – the alternative NHEJ (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). The goal of my thesis consisted in identifying and characterising major actors of this pathway. In the absence of KU, alternative NHEJ would be initiated by ssDNA resection of damaged ends. We showed that the nuclease activity of MRE11 is necessary for this mechanism. MRE11 overexpression leads to a two fold stimulation of NHEJ efficiency, while the extinction of MRE11 by siRNA results in a two fold decrease. Our results demonstrate that the proteins RAD50 and CtIP act in the same pathway as MRE11. Moreover, in cells deficient for XRCC4, MIRIN – an inhibitor of the MRN complex – leads to a decrease in repair efficiency, implicating MRE11 in alternative NHEJ. We also showed that MRE11 can act in an ATM-dependent and independent manner (Rass et Grabarz Nat Struct Mol Biol 2009). The initiation of break resection needs to be pursued by a more extensive degradation of DNA, which is accomplished in yeast by the proteins Exo1 and Sgs1/Dna2. In human cells, in vitro studies have recently proposed a similar model of a two-step break resection. We chose to elucidate the role of one of the human homologs of Sgs1 – the RecQ helicase BLM – in the resection process. Our experiments show, that he absence of BLM decreases the efficiency of end joining by NHEJ, accompanied by an increase in error-prone events, especially long-range deletions (>200nt). This suggests that BLM protects against extensive resection during alternative NHEJ. Furthermore, BLM is implicated in the protection against CtIP-dependent resection at the initiation of HR. In conclusion, our results show a major role of BLM in protecting against an excess of resection, mediated by the MRN cofactor – CtIP. BLM interacts with 53BP1 at sites of damage, in an ATM-dependent manner, in order to regulate the resection process and counteract BRCA1 activity. This underlines the novel role of BLM in the protection against resection and favouring gene conversion events without crossing-over, which is substantial for maintaining genomic integrity.

Background: Therapy resistance is a great challenge in cancer treatment. Among numerous factors, cell adhesion to extracellular matrix is a well-known determinant of radiochemo-resistance. It has been shown that targeting focal adhesion proteins (FAPs), e.g. β1 integrin, enhances tumor cell radio(chemo)sensitivity in various entities such as head and neck squamous cell carcinoma (HNSCC), lung carcinoma, glioblastoma, breast carcinoma and leukemia. Previous studies demonstrated a functional crosstalk between specific FAPs and DNA repair processes; however, the molecular circuitry underlying this crosstalk remains largely unsolved. Hence, this study in HNSCC aimed to identify alternative FAPs associated with DNA damage repair mechanisms and radioresistance. Materials and Methods: A novel 3D High Throughput RNAi Screen (3DHT-RNAi-S) using laminin-rich extracellular matrix (lrECM) was established to determine radiation-induced re-sidual DNA double strand breaks (DSBs; foci assay) and clonogenic radiation survival. In the screen, we used UTSCC15 HNSSC cells stably expressing the DSB marker protein 53BP1 tagged to pEGFP. Validations were performed in 10 additional HNSCC cell lines (Cal33, FaDu, SAS, UTSCC5, UTSCC8, UTSCC14, UTSCC15, UTSCC45 and XF354fl2) grown in 3D lrECM. Immunofluorescence staining, immunoblotting, chromatin fractionation were utilized to evaluate protein expression, dynamics and kinetics post irradiation. Investigations of molecular mechanisms of DNA repair and radio(chemo)resistance employed DSB repair reporter assays for non-homologous end joining (NHEJ) and homologous recombination (HR), cell cycle analysis, chromatin fractionation levels evaluation and kinase activity profiling (PamGene) upon protein knockdown in combination with/-out X-ray exposure. Foci assay and clonogenic survival assay were performed after single or multiple knockdowns of synemin and associated proteins such as DNA-PKcs and c-Abl. Protein-protein interactions between synemin and associated proteins were determined using immunoprecipitation and proximity ligation assay. Mutant/depletion constructs of synemin (ΔLink-Tail, ΔHead-Link, Synemin_301-961, Synemin_962-1565, S1114A and S1159A) were generated in order to identify essential synemin’s sites controlling DNA repair functions. Results: Among the targets found in the 3DHT-RNAi-S, synemin was one of the most promising FAP candidates to determine HNSCC cell survival and DNA damage repair. Synemin silencing radiosensitized HNSCC cells, while its exogenous overexpression induced radio-protection. Radiation induced an increased synemin/chromatin interaction and a marked ac-cumulation of synemin in the perinuclear area. Intriguingly, synemin depletion elicited a 40% reduction in NHEJ activity without affecting HR or Alt-EJ. In line, ATM, DNA-PKcs and c-Abl phosphorylation as well as Ku70 expression strongly declined in synemin depleted and irra-diated cells relative to controls, whereas an opposite effect was observed under synemin overexpression. Single, double and triple depletion of synemin, DNA-PKcs and c-Abl resulted in a similar radiosensitizing effect and DSB levels as detected upon single knockdown of synemin, describing its upstream role. In kinome analysis, tyrosine kinases showed signifi-cantly reduced activity after synemin silencing relative to controls. Furthermore, immunoprecipitation assays revealed a protein complex formed between synemin, DNA-PKcs and c-Abl under pre- and post-irradiation conditions. This protein complex dispersed when ATM was pharmacologically inhibited, implying synemin function to be dependent on ATM kinase activity. By means of the different mutation/deletion constructs of synemin, the phosphorylation site at serine 1114 located on the distal portion of synemin’s tail was identified as essential protein-protein interaction site for synemin’s function in DNA repair. Conclusions: The established 3DHT-RNAi-S provides a robust screening platform for identifying novel targets involved in therapy resistance. Based on this screen and detailed mechanistic analyses, the intermediate filament synemin was discovered as a novel important determinant of DNA repair, tyrosine kinase activity and radiochemoresistance of HNSCC cells. These results further support the notion that DNA repair is controlled by cooperative interactions between nuclear and cytoplasmic proteins.

DNA DSBs are most toxic to cells because they can lead to genomic rearrangements and even cell death. Most DSBs induced by ionizing radiation or radiomimetic drugs such as calicheamicin and bleomycin, bear 3′-phosphate or 3′- PG moieties that must be removed to allow subsequent gap filling and ligation. DSBs can be repaired by two main pathways: the homologous recombination (HR) pathway and the non-homologous end-joining (NHEJ) pathway, NHEJ is the primary repair pathway in mammalian cells. While HR repairs single strand breaks (SSBs) or DSBs accurately by using an undamaged copy of the sequence mostly at late S phase and G2 phase, the NHEJ pathway repairs DSBs without the requirement for sequence homology in a processing that may be error-free or error- prone and is most active at G1 phase. TDP1 is a DNA repair enzyme in both pathways, It associates with DNA SSB repair proteins XRCC1 and DNA ligase III and plays a role in processing of topoisomerase I- mediated SSBs. Our early results suggested that TDP1 also can remove protruding 3’- PG and other 3’ blocks from DSBs ends in vitro. A homozygous H493R mutation in the active site of TDP1 causes spinocerebellar ataxia with axonal neuropathy (SCAN1), a rare autosomal recessive genetic disease with neurological symptoms including peripheral neuropathy. DNA damage and misrepair can be determined by measuring the incidence of chromosomal aberrations such as rings, breaks, dicentrics, acentric fragments, and translocations in metaphase cells, and micronuclei in interphase cells. To assess the possible role of TDP1 in DSB repair in intact cells, the radiosensitivity of SCAN1 cells was determined by using a dose-fractionation method of irradiation. The data indicated that, when exposed to fractionated radiation doses, the SCAN1 cells were more sensitive than normal cells. Moreover, following treatment of cells with calicheamicin, SCAN1 cells showed a significantly higher incidence of dicentric chromosomes, acentric fragments, and micronuclei compared to normal cells, indicating that calicheamicin-induced DSBs were repaired less accurately and less efficiently, or more slowly in SCAN1 cells than in normal cells. All these results are consistent with a role for TDP1 in repair of 3’-PG DSBs in vivo. Oxidative stress is thought to induce replicative senescence and DNA damage in mouse embryo fibroblasts (MEFs). To determine the possible roles of oxidative stress on Tdp1-deficient MEFs, Tdp1-knockout MEFs and normal MEFs were cultured in 20% oxygen (atmospheric) and 3% (physiological) oxygen. The data from growth assays indicated that normal MEFs showed replicative senescence in 20% oxygen but not in 3% oxygen. Tdp1-knockout MEFs showed very poor growth compared to Tdp1 normal MEFs in both oxygen conditions, clearly suggesting an influence of repair of Tdp1 on oxidative stress induced DNA-DSBs in MEFs. Taken together, our results indicated that TDP1 is capable of removing protruding 3’-PG from DSB ends in intact cells. Moreover, DSBs induced by oxidative stress were repaired more slowly or inefficiently in MEFs when Tdp1 is absent, resulting in cell cycle arrest and poor cell growth.

This thesis investigates two separate features of nonhomologous end-joining (NHEJ) DNA repair: end processing, and DNA repair kinase signaling. DNA end processing was investigated in a mouse model of hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), a congenital neurodegenerative disease. SCAN1 is caused by a homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1). To address how the H493R mutation elicits the specific pathologies of SCAN1 and to further elucidate the role of TDP1 in processing DNA end modifications, we generated a Tdp1 knockout mouse and characterized their behavior and specific repair deficiencies in extracts of embryonic fibroblasts from these animals. While Tdp1(-/-) mice appear phenotypically normal, extracts from Tdp1(-/-) fibroblasts exhibited deficiencies in processing 3'-phosphotyrosyl single-strand breaks and 3'-phosphoglycolate (PG) double-strand breaks (DSBs). Supplementing Tdp1(-/-) extracts with H493R TDP1 partially restored processing of 3'-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on PG termini on 3' overhangs of DSBs; these remained completely unprocessed. Altogether, these results suggest that for 3'-PG overhang lesions, the SCAN1 mutation confers loss of function, while for 3'-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate. Furthermore, there is evidence that TDP1 also localizes to mitochondria, and mitochondrial DNA damage should not be excluded from significantly contributing to SCAN1 pathology. The effect of ATM signaling on NHEJ was investigated via a novel vector that allows for inducing I-SceI-mediated DNA DSBs that can then be analyzed for NHEJ repair events by fluorescence- and PCR-based methods. Using highly specific DNA kinase inhibitors and the repair cassette, we showed that inhibiting ATM reduced NHEJ by 80% in a U87 glioma model. Analysis of the PCR products from the NHEJ repair vector by PsiI restriction cleavage allowed for assessment of the fidelity of the NHEJ repair: inhibiting ATM reduced high-fidelity NHEJ by 40%. Together, these results suggest that ATM is critical for NHEJ of I-SceI DSBs and for high-fidelity repair, possibly due to ATM's effects on chromatin architecture surrounding the DSB.

DNA double-strand breaks (DSBs) containing unligatable termini are potent cytotoxic lesions leading to growth arrest or cell death. The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3′-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3′-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. Moreover, Polynucleotide Kinase/ Phosphatase (PNKP) is known to process 3′-phosphates and 5′-hydroxyls during DSB repair. PNKP-deficiency sensitized both HCT116 and HeLa cells to 3′-phosphate ended DSBs formed upon radiation and radiomimetic drug treatment. The increased cytotoxicity in the absence of PNKP was synonymous with persistent, un-rejoined 3′-phosphate-ended DSBs. However, DNA-PK deficiency sensitized PNKP-/- cells to low doses of NCS suggesting that, in the absence of PNKP, alternative enzyme(s) can remove 3′-phosphates in a DNA-PK-dependent manner. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining. In addition, loss of PNKP significantly sensitizes cells to 3′-phosphate-ended DSBs due to a defect in 3′-dephosphorylation.

Ionizing radiation induces a range of different DNA lesions. In terms of mutation frequency and mammalian cell survival, the most critical of these lesions is the DNA double-strand break (DSB). DSB left unrepaired or mis-repaired may result in chromosomal aberrations that can lead to permanent genetic changes or cell death. The complexity of the DNA damage and the capacity to repair the DSB will determine the fate of the cell. This thesis focuses on the DNA damage formation, repair and signaling after irradiation of human cells. Radiation with high linear energy transfer (LET) produces clustered damaged sites in the DNA that are difficult for the cell to repair. Within these clustered sites, non-DSB lesions are formed that can be converted into a DSB and add to the damage complexity and affect DSB repair and the measurement. Heat-labile sites in DNA are converted into DSB at elevated temperatures. We show that heat-released DSB are formed post-irradiation with high-LET ions and increase the initial yield of DSB by 30%-40%, which is similar to yields induced by low-LET radiation. DNA-PKcs, a central player in non-homologous end-joining (NHEJ), the major mammalian DSB repair pathway, has been found to be both up- and downregulated in different tumor types. In Paper II we show that low levels of DNA-PKcs lead to extreme radiosensitivity but, surprisingly, had no effect on the DSB repair. However, the fraction of cells in G2/M phase increased two-fold in cells with low levels of DNA-PKcs. The study continued in Paper IV, where cells were synchronized to unmask potential roles of DNA-PKcs in specific cell cycle phases. Irradiation of DNA-PKcs suppressed cells in the G1/S phase caused a delay in cell cycle progression and an increase in accumulation of G2 cells. Further, these cells showed defects in DNA repair, where a significant amount of 53BP1 foci remained after 72 h. This further strengthens the hypothesis that DNA-PKcs has a role in regulation of mitotic progression. Several cellular signaling pathways are initiated in response to radiation. One of these downstream signaling proteins is AKT. We identified an interaction between DNA-PKcs and AKT. Knockouts of both AKT1 and AKT2 impaired DSB rejoining after radiation and low levels of DNA-PKcs increased radiosensitivity and decreased DNA repair further.

Les cassures double-brin (CDBs) de l'ADN sont décrites comme étant les lésions de l'ADN les plus délétères puisqu'elles conduisent systématiquement à la mort de la cellule si elles ne sont pas réparées. Les CDBs peuvent être réparées par différents mécanismes et notamment par Non-Homologous End-Joining (NHEJ). Chez les eucaryotes, les protéines centrales de la NHEJ, Ku70 et Ku80, forment un hétérodimère capable de se lier aux extrémités de l'ADN générées par la cassure. Par la suite, Ku70 et Ku80 recrutent de nombreuses autres protéines permettant la modification des extrémités et la réparation de la CDB par ligation. La NHEJ a également été caractérisée chez un nombre limité de bactéries chez qui le mécanisme semble moins complexe que chez les eucaryotes. Chez les bactéries, la NHEJ nécessite seulement deux protéines : un homodimère de Ku, et la protéine multifonctionnelle LigD capable de modifier les extrémités et d'effectuer la ligation. La majorité des études faites sur la NHEJ ont été menées chez des bactéries ne possédant qu'une seule paire des gènes ku/ligD. Cependant, de nombreux autres génomes bactériens possèdent plusieurs copies de ces deux gènes et le fonctionnement de la NHEJ chez ces organismes est inconnu. Le génome de la bactérie symbiotique Sinorhizobium meliloti code quatre Ku putatives (ku1-4) et quatre LigD putatives (ligD1-4). A ce jour, une seule étude a été menée chez ce modèle bactérien montrant que chacun des simples mutants ku est plus sensible que la souche sauvage à un traitement aux rayonnements ionisants, suggérant que chacune des Ku joue un rôle dans la réparation des CDBs par NHEJ. Par l'utilisation de différentes approches in vivo, nous avons mené une caractérisation génétique de la NHEJ chez S. meliloti permettant de clarifier les contributions relatives des gènes ku et ligD dans le mécanisme. Pour la première fois chez une bactérie, nous avons pu obtenir des résultats montrant la présence de plusieurs systèmes indépendants de NHEJ chez S. meliloti, et suggérant l'existence d'un possible hétérodimère de Ku. Nous avons également mis en évidence que la NHEJ est activée dans différentes conditions de stress, telles que le stress thermique et la carence nutritive, et qu'une partie de cette réparation est sous le contrôle du régulateur central de la réponse générale au stress RpoE2. Par ailleurs, nous avons montré que la NHEJ, et plus généralement les mécanismes de réparation des CDBs sont impliqués dans la résistance à la dessiccation chez S. meliloti. Enfin, nous avons généré la première preuve expérimentale d'une implication de la NHEJ dans le transfert horizontal de gène chez les bactéries. Dans leur ensemble, ces travaux enrichissent nos connaissances sur les mécanismes de réparation des CDBs chez les bactéries possédant plusieurs orthologues de Ku et LigD. Ils suggèrent également que la NHEJ pourrait contribuer à l'évolution des génomes, en particulier en condition de stress, non seulement en raison du caractère mutagène de ce type de réparation mais également en participant à l'acquisition d'ADN exogène originaire de bactéries distantes. / DNA double-strand breaks (DSBs) are described as the most deleterious DNA damages as they can lead to cell death if they are not repaired. DSBs can be repaired through several mechanisms, including Non-Homologous End-Joining (NHEJ). In eukaryotes, the main NHEJ proteins, Ku70 and Ku80, bind DNA ends as a heterodimer, and then recruit several additional proteins including enzymes which catalyze the processing and ligation of DNA ends. NHEJ has also been characterized in a limited number of bacteria, where the repair mechanism appears to be less complex than in eukaryotes. Indeed, only two proteins are required: a homodimeric Ku protein, and a multifunctional LigD enzyme able to process and ligate the DNA ends. However, most studies were performed on bacterial species encoding a single pair of ku/ligD. Actually, many bacterial species encode multiple copies of these genes, whose relative contributions to NHEJ in vivo are so far unknown. The Sinorhizobium meliloti genome encodes four putative Ku (ku1-4) and four putative LigD (ligD1-4). To date, a single study conducted on this model bacterium showed that every ku single mutant is more sensitive than the wild type strain to ionizing radiations showing that all ku genes are involved in NHEJ repair of DSBs in this organism. Here, using several in vivo approaches, we performed a comprehensive genetic characterization of NHEJ repair in S. meliloti, and clarified the respective contributions of the various ku and ligD genes. For the first time in bacteria, we obtained results showing the presence of several independent NHEJ systems in S. meliloti and suggesting the existence of a putative heterodimeric form of Ku. We also demonstrated that NHEJ repair is activated under various stress conditions, including heat and nutrient starvation, and that part of this repair is under the control of the general stress response regulator RpoE2. We showed that NHEJ and more generally DSB repair mechanisms are involved in desiccation resistance in S. meliloti. Finally, for the first time in bacteria, we provided evidence that NHEJ not only repairs DSBs, but can also erroneously integrate heterologous DNA molecules into the breaks. Altogether, our data provide new insights into the mechanisms of DSB repair in bacteria which encode multiple Ku and LigD orthologues. It also suggest that NHEJ might contribute to the evolution of bacterial genomes under adverse environmental conditions not only through error-prone repair of DSB by its mutagenesis repair characteristic but also by participating in the acquisition of foreign DNA from distantly related organisms during horizontal gene transfer events.