Rad51 paralogues Rad55–Rad57 balance the antirecombinase Srs2 in Rad51 filament formation

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions inDNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion. Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined. Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55–Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55–Rad57 heterodimer associates with the Rad51–single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51–Rad55–Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55–Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55–Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease. Rad51 protein and its homologues RecA, UvsX and RadA form nucleoprotein filaments with ssDNA that perform homology search and DNA strand invasion during homologous recombination. The Rad51 paralogues share the RecA core with the Rad51 protein featuring unique aminoand carboxy-terminal extensions (Supplementary Fig. 2), but themselves do not form filaments and are unable to perform homology search andDNA strand invasion. Whereas humans contain five paralogues (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3), the budding yeast Saccharomyces cerevisiae contains two clearly identifiable paralogues, Rad55 and Rad57 (Supplementary Fig. 2). Rad55 and Rad57 in yeast as well as the five human RAD51 paralogues have unique non-redundant functions in recombination, and mutations in any one of them lead to recombination defects, chromosomal instability, sensitivity to DNA damage, and meiotic defects. Defects in the budding yeast RAD55 and RAD57 genes lead to identical and epistatic phenotypes in DNA repair and recombination, consistent with the formation of a stable Rad55–Rad57 heterodimer. Rad55–Rad57 heterodimers were inferred to function as mediator proteins allowing assembly of the Rad51 nucleoprotein filament on ssDNA covered by the eukaryotic ssDNA-binding protein RPA. This suggested that Rad55–Rad57 are involved in the nucleation of the Rad51 filament, which is otherwise inhibited on RPA-covered ssDNA.This nucleationmodel is akin to the role of RecFORor BRCA2 in nucleating RecA or human RAD51 filaments. Rad51 filament formation in vivo can be monitored cytologically as Rad51 focus formation at the site of DNA damage. Unexpectedly, Rad51 focus formation after ionizing radiation in yeast was demonstrated to be independent of Rad55–Rad57 and formation of visible Rad55– Rad57 foci required Rad51 (ref. 10). These results are difficult to reconcile with the nucleation model derived from the biochemical results and suggest an alternative function of Rad55–Rad57 in vivo. To address the function of the Rad51 paralogues in yeast, we determined the effect of Rad55–Rad57 on the stability of Rad51–ssDNA nucleoprotein complexes. Deletion mutants of the RAD55 or RAD57 genes display a curious enhancement of some phenotypes at low temperature (in particular ionizing radiation sensitivity; see Supplementary Fig. 12), indicating that these proteins are involved in the stabilization of a molecular complex, probably the Rad51 presynaptic filament. To test this hypothesis, we incubated subsaturating amounts of Rad51 proteinwith ssDNA (1Rad51 per 15 nucleotides) in the presence of substoichiometric amounts of Rad55–Rad57 heterodimer (1Rad55–Rad57per 4Rad51) and challenged the filamentswith buffer containing a high salt concentration (500mM NaCl) (Supplementary Fig. 3a, b). Under these conditions, Rad51 does not maintain stable complexes with ssDNA during electrophoresis. However, the presence of Rad55–Rad57 resulted in stable, Rad51-containing ssDNA complexes that withstood the salt challenge. In a complementary approach, we examined the effect of Rad55–Rad57 on Rad51 filament formation at near-physiological ionic strength (90mM NaCl) (Fig. 1a, b). Under these conditions, only a fraction of the available Rad51 binds ssDNA, causing retarded mobility of the DNA (Fig. 1b, lane 3). Addition of substoichiometric amounts of Rad55–Rad57 (1 Rad55–Rad57 per 6 Rad51 in lane 4 of Fig. 1b) led to the formation of a novel, supershifted complex that contained both Rad51 and Rad55–Rad57, as demonstrated by immunoblotting. Rad55–Rad57 heterodimer alone binds to DNA under these conditions, leading to the formation of protein networks that are too large to enter the gel (Fig. 1b, lane 2). The results from both experiments (Fig. 1b; Supplementary Fig. 3) indicate that Rad55–Rad57 forma co-complex with Rad51 on ssDNA and stabilize Rad51–ssDNA filaments. Indeed, immunogold electronmicroscopy targeted towards Rad55 (glutathione S-transferase (GST)-tag; see Fig. 1c) directly visualized Rad55 associated with the Rad51–ssDNA filaments (Fig. 1d). Control experiments demonstrated the specificity of the gold labelling (Supplementary Table 1) with over 90% of the gold particles associated with clearly identifiable Rad51 filaments. The remainder may have associated with filaments too short to be scored or with free Rad55–Rad57. Gold particles were found either at the filament terminus (n5 40) or