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Dynamique de la chromatine et la régulation du changement du type sexuel chez la levure

Abstract : Homologous recombination (HR) is a DNA repair pathway dedicated to double-stranded DNA breaks (DSB) that is essential for genome integrity. In yeast Saccharomyces cerevisiae, study of the "mating type switch" as a model of HR has helped to understand several aspects of this repair pathway. The mating type switching involves the allelic conversion of the MAT locus from a "MATa" form to "MATα". This mechanism is initiated by the appearance of a single DSB induced by the HO endonuclease at the MAT locus. The DSB is then repaired by HR with one of the homologous sequences, HMLα and HMRa, present on chromosome III. The recurrent choice of the donor of the opposite sexual type ensures the allelic change of the MAT locus. Thus, the study of the directionality of donor selection allowed the identification of a sequence called the "recombination enhancer" (RE) located near the HMLα locus on the left arm of chromosome III. In MATa cells, the "activated" RE would allow the formation of a loop between the RE region and the MAT locus promoting efficient recombination with the close sequence HMLα. However, the formation of this loop on chromosome III and the factors involved are still poorly characterized. During my thesis work, I studied the organization of chromosome III according to the sexual type of yeast S. cerevisiae before the induction of HO endonuclease and HR events. My working hypothesis suggests that the RE would play a role on the organization of chromosome III before the appearance of DSB; contributing to the increased efficacy of donor selection during mating type switching. Thus, I was able to highlight two distinct roles for the left and right part of the RE. The left side of the RE is involved in donor selection during HR events but has a moderate effect on chromosome III folding when removed. The right part of the RE seems indispensable for the organization of chromosome III in both sexual types MATa and MATα. These results suggest that the modulation of chromosome organization via an enhancer sequence could contribute to donor selection in HR. In a second time, my thesis work was directed towards the understanding of the events of repair of a DSB. I used the advantage of the mating type switch model to induce a single DSB in the genome via the HO endonuclease. This approach enabled me to set up a study to identify the factors recruited at the repair site in order to better understand the dynamics of the events involved in HR. Indeed, the precise regulation of activity as well as the addressing of key repair factors at the fracture level have still not been clearly characterized. The main current challenges remain in the identification of all the players present at a single DSB site at a given moment. In this context I studied the repair dynamics of a DSB by co-immunoprecipitating the repair factors with the help of the ANCHOR-FLAG system inserted near the MAT fracture site. Co-immunoprecipitation kinetics during DSB induction allowed me to study the sequential arrival of proteins involved in repair. Analysis of the samples by mass spectrometry reveals more than twenty proteins involved in the repair. These preliminary results are very encouraging. The development of this approach will better characterize the dynamics of DNA repair events by HR. The ChIP-MS system via ANCHOR allows for the first time a proteome analysis on a single break site.
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Sylvain Audibert. Dynamique de la chromatine et la régulation du changement du type sexuel chez la levure. Biologie de la reproduction. Université Paul Sabatier - Toulouse III, 2017. Français. ⟨NNT : 2017TOU30396⟩. ⟨tel-02484775⟩



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