Étude de l'association entre le complexe Smc5/6 et les microtubules sur la chromatine péricentromérique

Chromosome folding has recently emerged as an essential factor in maintaining genome integrity. In yeast, chromosomes follow a Rabl configuration that plays an important role in maintaining genome integrity in the event of DNA damage. However, how dynamic chromosome folding is controlled and its role in the repair of double-strand breaks (DSBs) is poorly understood. Among the factors regulating chromosome folding, the conserved family of SMC proteins, which includes cohesins, condensins and Smc5/6, plays an essential role. The yeast Smc5/6 complex is enriched at the pericentromere, recruited during DSBs, interacts with microtubules and forms DNA loops by extrusion. In this study, we investigate the role of the Smc5/6 complex and its interaction with microtubules by engineering a mutant of the Smc5/6 complex, in which two lysines (K624 and K631) of the Smc5 protein are replaced by glutamic acid. We show that these mutations reduce microtubule binding in vitro. To define the effects of the smc5-2KE mutant on chromatin, we monitored the dynamics of several chromosomal loci in vivo at high temporal resolution. Our results show that, in the absence of DNA damage, the smc5-2KE mutation increases chromatin mobility along the chromosome. In regions far from the centromere, microtubule depolymerization or kinetochore disruption mimics the effects of the smc5-2KE mutant. In the pericentromeric region, the smc5-2KE mutation increases chromatin dynamics through both microtubule release and chromatin structural changes, as confirmed by polymer modeling. We investigated the role of the smc5-2KE mutant in DNA damage repair by inducing DSB at a position near or far from the centromere. We found that DSB repair by homologous recombination is significantly reduced in the smc5-2KE mutant only at the pericentromeric position. Finally, the smc5-2KE mutant has an impact on the mitotic spindle, centromere clustering and cytokinesis. Overall, our results show a specific effect of microtubule association with Smc5/6 for functional folding of pericentromeric chromatin to ensure genomic stability.