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

Abstract

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA 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.

Figure 1. Rad55-Rad57 is associated with and stabilizes Rad51-ssDNA filaments

a, Rad51-ssDNA filament assembly assay. b, 0.67 μM Rad51 ± 0.11 μM Rad55-Rad57 was incubated with 4 μM φX174 ssDNA. The migration position of free protein was confirmed in controls lacking DNA (Supplementary Fig. 3c). c, Reaction scheme of immunoaffinity gold labeling of Rad55. d, EM images of gold-labeled Rad55 associated with Rad51-ssDNA filament (1:3 Rad51/nucleotide; 2.34 μM Rad51 ±0.43 μM Rad55-Rad57, 7 μM (nt) ssDNA). Scale bars: 100 nm. e, Models for the disposition of Rad55-Rad57 with the Rad51 filament. For simplicity, only model 2 is drawn in all illustrations.

Figure 2. Rad55-Rad57 stabilizes Rad51-ssDNA filaments to resist disruption by Srs2

a, Pulldown assay measuring stability of Rad51-ssDNA complexes (1 Rad51: 3 nt, 1 μM Rad51 ± 0.1 μM Rad55-Rad57) against disruption by Srs2 (0.1 or 0.33 μM). b, Rad51 remaining bound to ssDNA. c, Quantitation of results in (b) and additional experiments. Shown are means ± 1 sd, n=3. d, Concomitant binding of Rad55-Rad57 and Srs2 to Rad51-covered ssDNA. Pulldown assay measuring stability of Rad51-ssDNA complexes (1 Rad51: 3 nt, 1 μM Rad51 ± 0.2 μM Rad55-Rad57) against disruption by 0.33 μM Srs2. Top, pulldowns; bottom, supernatants.

Figure 3. Rad55-Rad57 inhibit disruption of Rad51 presynaptic filaments by Srs2

a, RPA-ssDNA complex. b, Short (145 nm) Rad51-ssDNA filament. c, Long (350 nm) Rad51-ssDNA filaments d, Quantitation of electron microscopic analysis. 300-400 filaments were analyzed for each reaction condition (2.34 μM Rad51, 7 μM (nt) 600 nt ssDNA, ± 0.43 μM Rad55-Rad57, ± 0.21 μM RPA, ± 0.4 μM Srs2), and the means (ø) ± 1 sd and distributions of filament length classes are shown. Scale bars: 100 nm. White arrows indicate RPA-ssDNA complexes and red arrows Rad51 filaments.

Figure 4. Rad55-Rad57 interact with Srs2 and inhibit Srs2 helicase

a, Pull-down with 4 nM (1 pmol) Rad55-Rad57 and 2.7, 8, or 16 nM Srs2. b, Pull-down with 4 nM Rad55-Rad57 and 8 or 16 nM Rad51. c, Quantitation of results in (a) and (b) and additional experiments. d, Pull-down with 4 nM Rad55-Rad57 and 8 nM Srs2 ± 40 nM Rad51. GST was used as control. S: supernatant, W: wash, E: eluate. e, Helicase assay. f, 28 nM Rad51 ± 25 nM Rad55-Rad57 were incubated with 1.5 nM 3′-tailed substrate before addition of 120 nM Srs2 protein. Product yields at 20 min. were quantified as shown in g. HEAT DEN.: heat denatured substrate, shown are means ± 1sd, n=3.