Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

• Klíčová slova
• Genetics, Molecular biology, Molecular interaction, Properties of biomolecules,
• Publikační typ
• časopisecké články MeSH

Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group. The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex. We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact. This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex. Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding. Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process.

• MeSH
• DNA opravný a rekombinační protein Rad52 MeSH
• DNA primery genetika   MeSH
• DNA vazebné proteiny chemie   genetika   metabolismus   MeSH
• DNA-helikasy MeSH
• enzymy opravy DNA MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• genetická epistáze MeSH
• geny hub MeSH
• methylmethansulfonát toxicita   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• oprava DNA genetika   MeSH
• rekombinace genetická * MeSH
• rekombinasa Rad51 MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• Saccharomyces cerevisiae účinky léků   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• techniky dvojhybridového systému MeSH
• teplota MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• odvolaná publikace MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA opravný a rekombinační protein Rad52 MeSH
• DNA primery MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• enzymy opravy DNA MeSH
• fungální proteiny MeSH
• methylmethansulfonát MeSH
• RAD51 protein, S cerevisiae MeSH Prohlížeč
• RAD52 protein, S cerevisiae MeSH Prohlížeč
• RAD54 protein, S cerevisiae MeSH Prohlížeč
• RAD55 protein, S cerevisiae MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• Saccharomyces cerevisiae - proteiny * MeSH