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Crible protéomique pour identifier de nouvelles protéines associées à la recombinaison méiotique

proposé par Valérie BORDE, Institut Curie – UMR3244 « Dynamique de l’Information Génétique »

Projet de stage : DNA double strand breaks (DSBs) are the most dangerous DNA lesions for genome integrity. They can occur accidentally, or be part of a developmental program, such as during meiosis. During DSB repair by homologous recombination, a step of DNA synthesis is required to reconstitute the DNA degraded during DSB end resection. This essential step is poorly characterized, and in particular which factors are important for Recombination-Associated DNA Synthesis (RADS), how the extent of DNA synthesis is regulated and what are the consequences of its deregulation. Recently, our lab has identified a novel regulatory step of meiotic recombination, which controls the extent of DNA synthesis occurring during the repair of meiotic DSBs (Duroc et al. 2017). To identify new factors associated with RADS, the student will use a method recently optimized in the lab to monitor proteins associated with newly synthesized DNA in budding yeast during meiotic recombination. For this, budding yeast cells undergoing synchronous meiosis are incubated with a modified nucleotide, fixed and RADS-associated pulled-down proteins identified by either Western blot for a candidate-based approach or by mass spectrometry. We will identify and compare RADS proteins during meiotic and somatic recombination and in several mutant conditions. We expect with this unbiased approach to identify new factors present at DSB being repaired by homologous recombination, including DNA polymerases and their co-factors, but also proteins involved in signaling, in chromatin modifications or remodeling, or other DNA-based processes. Techniques mises en œuvre par le stagiaire : The project will employ genetic, molecular biology and in vivo protein analyses, using the budding yeast as a model system.

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