Localization to the nuclear pore complex is required for stabilizing CAG repeats

Abstract

CAG repeats are structure-forming repetitive DNA sequences which can interfere with DNA replication and DNA repair. CAG repeats are prone to breakage, and repair of the breaks can cause repeat expansions or contractions. Expansion beyond a threshold of ~35 CAG repeats is the cause of more than 14 human diseases including Huntington's disease and myotonic dystrophy. Studies in yeast have shown that irreparable DNA double strand breaks or collapsed replication forks localize to the nuclear periphery to promote proper repair. We tagged an expanded CAG repeat locus with GFP (green fluorescent protein) on a yeast chromosome, and visualized the localization of the repeats in the nucleus by using fluorescent microscopy. Our data showed that expanded CAG-70 and -130 repeats are more likely than a control to localize to the nuclear periphery during S-phase, in a tract length dependent manner. Through a mobility analysis, we also showed that the mobility of expanded CAG repeats decreases due to this localization. By chromatin immunoprecipitation (ChIP), we found that expanded CAG repeats interact with the Nup84 component of the nuclear pore complex. Moreover, Nup84 and its associated SUMO targeting ubiquitin ligase (STUbL), Slx5/Slx8, are important for preventing CAG repeat instability and fragility. Our results indicate that lesions associated with structure-forming repeats trigger a repair or fork restart event that occurs at the nuclear periphery, and that this event is crucial for the maintenance of genome stability. This study was funded by Tufts University.