The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.

• MeSH
• DNA metabolismus   MeSH
• endonukleasy chemie   genetika   metabolismus   MeSH
• enzymy opravy DNA chemie   genetika   metabolismus   MeSH
• intracelulární signální peptidy a proteiny fyziologie   MeSH
• lysin metabolismus   MeSH
• mutace MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• protein-serin-threoninkinasy fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   fyziologie   MeSH
• sumoylace * MeSH
• ubikvitinligasy fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH