Homotypic and heterotypic protein associations control Rad51 function in double-strand break repair

Abstract

Rad51 is essential for efficient repair of DNA double-strand breaks (DSBs) and recombination in Saccharomyces cerevisiae. Here, we examine Rad51 protein-protein interactions and their biological significance. GAL4 two- hybrid fusion analysis demonstrated that the amino-terminal region of Rad51 mediates both a strong Rad51:Rad51 self-association and a Rad51:Rad52 interaction. Several Rad51 variants were characterized that imparted DSB repair defects; these defects appear to result from Rad51 protein-protein interactions. First, a rad51 allele bearing a missense mutation in the consensus ATP-binding sequence disrupted DSB repair in wild-type yeast. The effect of this allele was dependent on the presence of wild-type Rad51 because MMS sensitivity of rad51Δ strains were not increased by its expression. Second, we identified a highly conserved RAD51 homolog from Kluyveromyces lactis (KlRAD51) that only partially complemented rad51Δ strains and impaired DSB repair in wild-type S. cerevisiae. Third, fusions of Gal4 domains to Rad51 disrupted DSB repair in a manner that required the presence of either Rad51 or Rad52. Because K. lactis RAD51 and RAD52 did not complement a S. cerevisiae rad51Δ rad52Δ strain, Rad51-Rad52 functions appear to be mediated through additional components. Thus, multiple types of Rad51 protein interactions, including self-association, appear to be important for DSB repair.