RESUMO
The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneous recombination that is not essential for DSB repair. Furthermore, we investigated the currently unknown mechanism of rad57 suppression by MAT heterozygosity and found that it is independent of DNL4.
Assuntos
Adenosina Trifosfatases/genética , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Reparo do DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/fisiologia , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo/genética , Conversão Gênica/genética , Genes Fúngicos Tipo Acasalamento/fisiologia , Heterozigoto , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Organismos Geneticamente Modificados , Multimerização Proteica , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Rad51 requires a number of other proteins, including the Rad51 paralogs, for efficient recombination in vivo. Current evidence suggests that the yeast Rad51 paralogs, Rad55 and Rad57, are important in formation or stabilization of the Rad51 nucleoprotein filament. To gain further insights into the function of the Rad51 paralogs, reporters were designed to measure spontaneous or double-strand break (DSB)-induced sister or nonsister recombination. Spontaneous sister chromatid recombination (SCR) was reduced 6000-fold in the rad57 mutant, significantly more than in the rad51 mutant. Although the DSB-induced recombination defect of rad57 was suppressed by overexpression of Rad51, elevated temperature, or expression of both mating-type alleles, the rad57 defect in spontaneous SCR was not strongly suppressed by these same factors. In addition, the UV sensitivity of the rad57 mutant was not strongly suppressed by MAT heterozygosity, even though Rad51 foci were restored under these conditions. This lack of suppression suggests that Rad55 and Rad57 have different roles in the recombinational repair of stalled replication forks compared with DSB repair. Furthermore, these data suggest that most spontaneous SCR initiates from single-stranded gaps formed at stalled replication forks rather than DSBs.
