Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52.
Saccharomyces cerevisiae rad52 mutants are characterized by severe defects in double-strand break (DSB) repair and recombination. In this study we have identified several regions of RAD52 that are required for these biological functions. We cloned and characterized a RAD52 homolog from Kluyveromyces lactis that partially complemented S. cerevisiae rad52 mutants while exhibiting negative dominance in wild-type (RAD52) strains. The dominant negative effect was suppressed by overexpression of RAD51, an additional gene known to be required for DSB repair and recombination, indicating a genetic interaction between these loci. Furthermore, GAL4 two-hybrid analysis revealed a physical interaction between Rad51 and the carboxy-terminal one-third of Rad52. Deletion alleles of rad52 (with or without the Rad51 association domain) also produced dominant negative defects, suggesting the disruption of repair through nonfunctional interactions with other DSB repair and recombination proteins. RAD51 relieved the negative dominance of each of these alleles either by competitive titration or functional activation of mutant or heterologous Rad52 proteins. These results demonstrate the importance of Rad52-Rad51 interactions and point to the formation of a higher order repair/recombination complex potentially containing other yet unidentified components.
Milne
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Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Weaver
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D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D004260:DNA Repair / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D005799:Genes, Dominant; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007716:Kluyveromyces / Q000235:genetics; D008938:Mitosis / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase