Swi5-Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation

Abstract

Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5-Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

Keywords: Rad51; Swi5–Sfr1; homologous recombination; single-molecule microscopy.

Fig. 1.

Mouse SWI5–SFR1 stimulates mRAD51 nucleoprotein filament assembly. (A) Schematic illustration of the RAD51 nucleoprotein assembly experiments. (B) Representative bead BM time courses of mRAD51 (0.8 μM) assembly on (dT)₁₃₅ DNA substrates without mS5S1 (Upper) or with 1.6 μM mS5S1 (Middle) or 1.6 μM mS5^FLS1 mutant (Lower). Gray bars correspond to the dead time when recombinase mixtures with 2 mM ATP were introduced. (C–E) Histograms of nucleation time (C), mean extension time (seconds per RAD51) (D), and bead BM increment (E) of mRAD51 assembling. All experiments were carried out at 2 mM ATP. Error bars of nucleation rate are the SD of the mean by bootstrapping 5,000 times, and error bars of extension time are 1 SEM. (F) mRAD51 (0.8 μM) was preincubated with various stoichiometric ratios of mS5S1, and the mixture was introduced into a reaction chamber containing surface-bound (dT)₁₃₅ gapped DNA. Nucleation rates are about constant (∼0.010 s⁻¹) when the [mS5S1]/[mRAD51] ratio is less than 1.625. The nucleation rates of mRAD51 increase and achieve maximum value (∼0.015 s⁻¹) when the ratio of [mS5S1]/[mRAD51] is larger than 2, suggesting that maximum nucleation stimulation occurs in the mixture of one mRAD51 and two mS5S1s. Individual nucleation rates were obtained based on maximum-likelihood estimation. (G) Bead BM increment of mRAD51 assembly on (dT)₁₃₅ DNA substrates in the presence of the indicated ratios of mS5S1 to mRAD51. Bead BM increment reflects the coverage of the RAD51 nucleoprotein filaments. mRAD51 forms longer and more stable filaments in the presence of mS5S1. All experiments were carried out at 2 mM ATP. Black open squares represent the nucleation rate of mRAD51 in the presence of the nonhydrolyzable ATP analog AMPPNP in the absence of mS5S1. Dashed lines are the mean, and the shaded regions span 2 SDs. Error bars of bead BM increment are 1 SEM.

Fig. 2.

Mouse mS5S1 reduces the nucleation unit of mRAD51 and increases ssDNA affinity. (A and B) RAD51 concentration dependence of filament nucleation obtained by TPM experiments. Power-law fitting to the observed nucleation rates suggests the nucleation unit of RAD51: 2.43 ± 0.46 for mRAD51 (A) and 1.67 ± 0.16 for the mRAD51–mS5S1 complex (B). Green diamonds in A are nucleation rates of mRAD51 in the presence of excess mS5^FLS1 mutant defective in stimulating mRAD51. mS5S1 and mS5^FLS1 are in twofold excess in A and B. (C) ssDNA length dependence of mRAD51 filament nucleation rate obtained by TPM experiments. Gapped DNA substrates contain only one 5′ ds/ss junction but various lengths of ssDNA gaps (90 to 200 nt). As the gapped DNA substrate structure, overall nucleation rates are fitted to k_ssDNA^app(L_ssDNA) + k_junction^app, where k_ssDNA^app and k_junction^app are apparent ssDNA-dependent nucleation rate constant and apparent ds/ss junction-dependent nucleation rate constant. Circle, 0.8 μM mRAD51 only; square, mixture of 0.8 μM mRAD51 and 1.6 μM mS5S1. All experiments were carried out at 2 mM ATP.

Fig. 3.

Single-molecule FRET experiments demonstrate that mS5S1 stabilizes mRAD51 nucleating clusters. (A) Schematic illustration of the single-molecule fluorescence resonance energy transfer experimental setup. mRAD51 assembling onto (dT)₁₃ ssDNA results in FRET decrease due to the increase of dye pair separation. (B–F) Single-molecule FRET observation of mRAD51 nucleating cluster dynamics. Exemplary FRET time traces of mRAD51 (B) and the mRAD51–mS5S1 complex (C) assembling on (dT)₁₃ ssDNA substrate. High-FRET state (∼0.8) corresponds to a DNA-only state, and low-FRET state (0.0∼0.6) corresponds to the mRAD51-bound state. (D and E) Transition density plots clearly reflect four states (without mS5S1) and five states (with mS5S1) in mRAD51 nucleating cluster dynamics. (F) Rate constants of mRAD51 nucleating cluster dynamics in the absence (empty bars) and presence (solid bars) of mS5S1. Error bars of binding and dissociation rates are the SD of the mean by bootstrapping 5,000 times. N.A., data not available.

Fig. 4.

Fission yeast SpS5S1 does not stimulate SpRad51 filament assembly. (A) Representative bead BM time courses of fission yeast Rad51 (0.8 μM) assembly on (dT)₁₃₅ DNA substrates without S5S1. (B–D) Nucleation time (B), mean extension time (C) (seconds per Rad51), and bead BM increment (D) analyzed from individual assembly time courses. Nucleation time histograms are fitted by single exponential decay. (E) Nucleation rates of various concentrations of SpS5S1 at constant 0.3 μM SpRad51. (F) Bead BM increments of SpRad51 assembly at various ratios of SpS5S1 and SpRad51 mixtures also decreased at higher SpS5S1 concentrations. All experiments were carried out at 2 mM ATP and 0.3 μM SpRad51. Gray solid circles are from wild-type SpS5S1 experiments. Black diamonds are from the N-terminal truncation mutant of SpS5S1 (SpS5S1C) deficient in ssDNA binding. Dashed lines are the mean, and the shaded regions span 2 SDs.

Fig. 5.

Nucleoprotein filament disassembly experiments showed that S5S1 prevents Rad51 filament disassembly. (A) Schematic illustration of nucleoprotein filament disassembly experiments using the TPM setup. (B) Representative bead BM time courses of mouse mRAD51 disassembly on (dT)₁₃₅ DNA substrates without mS5S1 (Upper) and in the presence of 1.0 μM mS5S1 (Lower). mRAD51 filaments were preassembled in the presence of ATP. Dark gray bars stand for void time for extensive buffer wash to remove free mRAD51. A lifetime of the preassembled filament before the BM decrease dictates the mRAD51 disassembly kinetics. (C–E) Mean lifetime of mouse mRAD51 nucleoprotein filament (C), fraction of the undisassembled filament within 15 min (D), and mean dissociation time per mRAD51 (E) in the presence of various mS5S1 concentrations and nucleotide conditions. (F) Representative bead BM time courses of SpRad51 disassembly without SpS5S1 (Upper) and in the presence of 0.3 μM SpS5S1 (Lower). (G–I) Kinetic parameters for fission yeast. The fraction of undisassembled tethers is correlated with the mean lifetime of the SpRad51 filament in both species. Here, the N-terminal truncation S5S1C mutants (open bars) are deficient in DNA binding. All experiments were carried out at 2 mM ATP. Error bars are 1 SEM.

Fig. 6.

Proposed models for regulating Rad51 nucleoprotein filament formation by the S5S1 complex. Swi5–Sfr1 stabilizes Rad51 on ssDNA primarily by preventing its dissociation. This stabilization effect leads to a stable nucleating cluster formation and a reduction in filament disassembly. Despite different kinetic properties of mouse and fission yeast Rad51, the Swi5–Sfr1 complex stimulates the Rad51 process through a general, evolutionarily conserved mechanism. Red half-arrows indicate the kinetic steps affected by S5S1.