Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

Abstract

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.

Figure 1.

Biophysical properties of the Swi5–Sfr1 complex. (A) Purified Swi5–Sfr1 (lane 1), Swi5 (lane 2), Sfr1 (lane 3), (His)₆-tagged Rad51 (His-Rad51, lane 4) and Strep-tagged Rad51 (Strep-Rad51, lane 5) were resolved by 13% SDS–PAGE and stained with Coomassie Blue staining. Note that Sfr1 (36 kDa) exhibits an aberrantly slow mobility in SDS–PAGE (17). (B) Isothermal titration calorimetry to determine the stoichiometric ratio of Swi5 to Sfr1. The top plot summarizes the programmed sequence of 17 injections of Swi5 into the chamber that contained Sfr1. The lower plot shows the integrated binding isotherm with experimental points and best fit. (C) Sedimentation equilibrium analysis of the Swi5–Sfr1 complex. The average molecular mass was estimated to be 47.5 kDa. The upper part of the figure shows the residual difference between experimental and fitted values by standard deviation.

Figure 2.

Promotion of Rad51-mediated DNA strand exchange by Swi5–Sfr1. (A) Schematic of the DNA strand exchange assay. The radiolabeled substrate and product are visualized and quantified by phosphorimaging analysis after PAGE. The asterisk denotes the ³²P label. (B) DNA strand exchange reactions with the indicated concentrations of Rad51 and Swi5–Sfr1. The incubation time was 20 min. (C) Time kinetics of DNA strand exchange mediated by Rad51 alone (1.6 µM) or with Swi5–Sfr1 (1.6 µM). The results were graphed. (D) DNA strand exchange by Rad51 (1.6 µM) with or without Swi5–Sfr1 (1.6 µM) was examined. Either ATP or ADP was included in the reaction, as indicated. The incubation time was 10 min. (E) The DNA strand exchange activity of RecA was examined and with or without the indicated concentration of Swi5–Sfr1. The incubation time was 20 min. (B–E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.

Figure 3.

Swi5–Sfr1 complex but not Swi5 or Sfr1 physically interacts with Rad51. (A) For affinity pulldown, purified Swi5–Sfr1 (S5S1) with a (His)₆ tag on Sfr1 was incubated with either Rad51 (panel I) or RecA (panel II), and the reaction mixtures were mixed with Talon beads to capture protein complex through the (His)₆ tag. The supernatant (S), wash (W) and SDS elute (E) from the pull-down reaction were analyzed by 13% SDS–PAGE with Coomassie Blue staining. Rad51 and RecA alone were included as controls. (B) Purified (His)₆-tagged Swi5, Sfr1 and Swi5–Sfr1 were incubated with Rad51 to examine their interaction. Symbol: S5S1, Swi5–Sfr1.

Figure 4.

Enhancement of Rad51-mediated DNA strand exchange by Swi5–Sfr1. (A) and (B) DNA strand exchange reactions containing Rad51 (1.6 µM) and the indicated amount of Swi5, Sfr1 or Swi5–Sfr1. The incubation time was 20 min. The results were graphed. Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.

Figure 5.

Stabilization of the Rad51 presynaptic filament by Swi5–Sfr1. (A) Schematic of the exonuclease I protection assay for examining presynaptic filament stability. The Rad51 presynaptic filament harboring 5′-³²P-labeled DNA is incubated with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The ³²P label is denoted by the asterisk. (B) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5–Sfr1. The results were graphed. (C) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5 (panel I), Sfr1 (panel II) or Swi5–Sfr1. The results were graphed. (B and C) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.

Figure 6.

Presynaptic filament stabilization by AMP-PNP or Ca²⁺ alleviates dependence on Swi5–Sfr1. (A) DNA strand exchange was conducted using AMP-PNP as the nucleotide cofactor. The results were graphed. (B) DNA strand exchange was conducted in the presence of Ca²⁺. The results were graphed. (A and B) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.

Figure 7.

Functional significance of the RSfp motif in Sfr1. (A) Sfr1 (303 residues) harbors a RSfp motif in its N-terminus. This motif is deleted in the dN104Sfr1 mutant. (B) Purified Swi5–dN104Sfr1 complex (1.5 µg) was subjected to 13% SDS–PAGE and Coomassie Blue staining. (C) Purified Swi5–dN104Sfr1 mutant complex was tested for Rad51 interaction by affinity pulldown through the (His)₆ tag on dN104Sfr1. The supernatant (S), wash (W) and SDS elute (E) from the pull-down reaction were analyzed by 13% SDS–PAGE with Coomassie Blue staining. (D) DNA strand exchange was conducted with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. (E) The stability of the Rad51 presynaptic filament was tested by the exonuclease I assay with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. (D and E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbols: S5S1, Swi5–Sfr1; S5-dN104S1, Swi5–dN104Sfr1.