Homotypic and heterotypic protein associations control Rad51 function in double-strand break repair.
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 rad51 delta strains were not increased by its expression. Second, we identified a highly conserved RAD51 homolog from Kluyveromyces lactis (KlRAD51) that only partially complemented rad51 delta 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 rad51 delta rad52 delta 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.
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts.
J W
JW
Donovan
G T
GT
Milne
D T
DT
Weaver
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005800:Genes, Fungal; D007700:Kinetics; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D051135:Rad51 Recombinase; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase