Um dos mais versáteis mecanismos de reparo de DNA é o reparo por excisão de nucleotídeos (nucleotide excision repair- NER). Defeitos genéticos associados a esta via podem gerar diferentes síndromes com deficiência de reparo. Dentre essas, Xeroderma pigmentosum (XP) é a que apresenta maior sensibilidade à luz solar, resultando em um grande aumento na incidência de tumores em regiões expostas da pele e, em alguns casos, degeneração neurológica progressiva e envelhecimento prematuro. Na primeira parte deste projeto é apresentado o uso de adenovírus recombinantes portando genes da via de NER para identificar a deficiência gênica de três pacientes portadores de XP. Na segunda parte do trabalho os estudos de reparo de DNA são estendidos a modelos animais, com deficiências nos mesmos genes carregados pelos vetores adenovirais. A expressão gênica do vetor foi avaliada pela detecção de proteína e por visualização da fluorescência de EGFP na pele dos animais infectados. Em resumo, este trabalho apresenta o uso eficiente de vetores adenovirais portando genes de reparo em ensaios in vitro e in vivo, e descreve duas mutações deletérias no gene XPC de pacientes XP brasileiros, incluindo uma mutação nova. / One of the most versatile mechanisms of DNA repair is the nucleotide excision repair (NER). Genetic defects in NER can generate different syndromes. Among these, Xeroderma pigmentosum) presents the highest sensitivity to sunlight, resulting in a large increase in the incidence of skin cancer, especially in areas exposed to the sunlight, and in some cases, progressive neurological degeneration and premature aging. In the first part of this project, adenoviral vectors carrying NER genes were used to identify genetic deficiency of three XP patients. The second part of work was extended to animal models, deficient for the same XP genes carried by adenoviral vectors. The genetic expression of vector was evaluated by detection of protein and EGFP fluorescence visualization in the skin of animals transduced. In summary, this work presents the use of adenovirus, carrying DNA repair genes for in vitro in vivo studies reports two deleterious mutations in Brazilian XP patients, including a new mutation.

Um dos mais versáteis mecanismos de reparo de DNA é o reparo por excisão de nucleotídeos (nucleotide excision repair- NER). Defeitos genéticos associados a esta via podem gerar diferentes síndromes com deficiência de reparo. Dentre essas, Xeroderma pigmentosum (XP) é a que apresenta maior sensibilidade à luz solar, resultando em um grande aumento na incidência de tumores em regiões expostas da pele e, em alguns casos, degeneração neurológica progressiva e envelhecimento prematuro. Na primeira parte deste projeto é apresentado o uso de adenovírus recombinantes portando genes da via de NER para identificar a deficiência gênica de três pacientes portadores de XP. Na segunda parte do trabalho os estudos de reparo de DNA são estendidos a modelos animais, com deficiências nos mesmos genes carregados pelos vetores adenovirais. A expressão gênica do vetor foi avaliada pela detecção de proteína e por visualização da fluorescência de EGFP na pele dos animais infectados. Em resumo, este trabalho apresenta o uso eficiente de vetores adenovirais portando genes de reparo em ensaios in vitro e in vivo, e descreve duas mutações deletérias no gene XPC de pacientes XP brasileiros, incluindo uma mutação nova. / One of the most versatile mechanisms of DNA repair is the nucleotide excision repair (NER). Genetic defects in NER can generate different syndromes. Among these, Xeroderma pigmentosum) presents the highest sensitivity to sunlight, resulting in a large increase in the incidence of skin cancer, especially in areas exposed to the sunlight, and in some cases, progressive neurological degeneration and premature aging. In the first part of this project, adenoviral vectors carrying NER genes were used to identify genetic deficiency of three XP patients. The second part of work was extended to animal models, deficient for the same XP genes carried by adenoviral vectors. The genetic expression of vector was evaluated by detection of protein and EGFP fluorescence visualization in the skin of animals transduced. In summary, this work presents the use of adenovirus, carrying DNA repair genes for in vitro in vivo studies reports two deleterious mutations in Brazilian XP patients, including a new mutation.

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.