Rad51, the major eukaryotic homologous recombinase, is important for the repair of DNA damage and the maintenance of genomic diversity and stability. The active form of this DNA-dependent ATPase is a helical filament within which the search for homology and strand exchange occurs. Here we present the crystal structure of a Saccharomyces cerevisiae Rad51 filament formed by a gain-of-function mutant. This filament has a longer pitch than that seen in crystals of Rad51's prokaryotic homolog RecA, and places the ATPase site directly at a new interface between protomers. Although the filament exhibits approximate six-fold symmetry, alternate protein-protein interfaces are slightly different, implying that the functional unit of Rad51 within the filament may be a dimer. Additionally, we show that mutation of His352, which lies at this new interface, markedly disrupts DNA binding.

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 DSBinduced 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.

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. H OMOLOGOUS recombination is required forthe faithful repair of DNA double-strand breaks (DSBs) that arise during normal cellular processes or from exposure of cells to DNA-damaging agents. Central to the process of homologous recombination is the Rad51 protein, which facilitates synapsis and strand invasion into homologous duplex DNA (San Filippo et al. 2008). Rad51 belongs to the RecA family of homologous pairing proteins (Aboussekhra et al. 1992;Basile et al. 1992;Shinohara et al. 1992). Yeast and humans have two RecA homologs: Rad51 and the meiosis-specific Dmc1 (Bishop et al. 1992;San Filippo et al. 2008). In addition, the Saccharomyces cerevisiae RAD55 and RAD57 genes encode proteins with sequence similarity to RecA and Rad51 and are considered to be Rad51 paralogs (Kans and Mortimer 1991;Lovett 1994). Mutation of RAD51, RAD55, or RAD57 confers sensitivity of ionizing radiation (IR) and defects in mitotic and meiotic recombination, indicating that their functions are not redundant (Symington 2002). rad51 mutants generally exhibit more severe defects than rad55 or rad57 mutants in DSB-induced recombination assays; however, rad55 and rad57 mutants are more defective than rad51 in some assays that measure spontaneous recombination between repeated sequences (Rattray and Symington 1995;Mozlin et al. 2008).The molecular details of homologous recombination are largely based on genetic, physical, and cytological studies of DSB repair (DSBR) and on biochemical characterization of purified proteins (Paques and Haber 1999;Sugawara et al. 2003;Lisby et al. 2004;San Filippo et al. 2008). The single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), initially binds ssDNA that forms by nucleolytic processing of DNA ends at DSBs. In vitro, RPA has been shown to be inhibitory to Rad51 binding to ssDNA, but this inhibition c...

The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. We suggest the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding.