Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination

Abstract

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.

Figure 1.

SWI5–SFR1 interacts with the oligomeric form of RAD51. For affinity pulldown, purified SWI5–SFR1 (S5S1) was incubated with either RAD51 wild-type (A) or RAD51 S208E/A209D (B) in the presence of an increasing concentration of the BRC4 peptide. Reaction mixtures were mixed with Talon resin to capture protein complex through the (His)₆ tag on SFR1. The supernatant (lanes 1–6) and SDS eluate (lanes 7–12) from the pulldown reactions were analyzed by 15% SDS-PAGE with Coomassie Blue staining. RAD51 alone was included as a control (lanes 1 & 7). (C) The results from (A) and (B) were quantified and plotted, and error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. (D) SWI5–SFR1 was incubated with RAD51 wild-type (WT), F86E, A190L/A192L, or S208E/A209D for affinity pulldown analyses. The supernatant (S), wash (W), and SDS eluate (E) fractions from the pulldown reactions were analyzed by 15% SDS-PAGE with Coomassie Blue staining. Symbols: R51, RAD51; R51^SA/ED, RAD51 S208E/A209D; S5S1, SWI5–SFR1.

Figure 2.

Functional characterization of SWI5–SFR1^dN202 complex. (A) To map the minimal complex unit of SWI5–SFR1, purified SWI5–SFR1 was incubated with proteinase K for the indicated times. The proteolytic products were resolved by 15% SDS-PAGE and stained by Coomassie Blue staining. The asterisk denotes the SFR1^dN202. (B) Purified SWI5–SFR1^dN202 complex (3 μg) was subjected to 15% SDS-PAGE and Coomassie Blue staining. (C) SWI5–SFR1^dN202 was tested for RAD51 interaction by affinity pulldown as described in Figure 1D. (D) DNA strand exchange assay to monitor the stimulatory effect of SWI5–SFR1 by RAD51. (I) 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. (II) DNA strand exchange was conducted with the indicated amounts of SWI5–SFR1 or SWI5–SFR1^dN202. The results were graphed. (E) Exonuclease I protection assay to monitor the influence of SWI5–SFR1 on the stability of RAD51 filament. (I) Schematic of the exonuclease I protection assay. The RAD51 filament harboring 5′-³²P-labeled DNA is challenged 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. (II) Treatment of the RAD51 presynaptic filament with exonuclease I in the presence of the indicated concentrations of SWI5–SFR1 or SWI5–SFR1^dN202. The results were graphed. (D-E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.

Figure 3.

SWI5^dC20–SFR1, SWI5^dC9–SFR1 and SWI5^FL/AA–SFR1 are defective in RAD51 interaction. (A) (I) Schematic of C-terminal truncation of SWI5 mutant variants made in this study. (II) Purified SWI5–SFR1, SWI5^dC20–SFR1 and SWI5^dC9–SFR1 were resolved by 15% SDS-PAGE and stained with Coomassie Blue. (B) Purified SWI5^dC20–SFR1 and SWI5^dC9–SFR1 mutant complexes were examined for RAD51 interaction by affinity pulldown through the (His)₆ tag on SFR1. Wild-type SWI5–SFR1 was included as positive control. (C) (I) Alignment of the C-terminal 9 amino acid residues of SWI5 from S. pombe, mouse, and human. (II) The SWI5–SFR1 mutants made in this study. (III) Purified SWI5 F83A/L85A–SFR1 (SWI5^FL/AA–SFR1), SWI5 F83A–SFR1 (SWI5^F/A–SFR1) and SWI5 L85A–SFR1 (SWI5^L/A–SFR1) were resolved by 15% SDS-PAGE and stained with Coomassie Blue. (D) Purified SWI5^FL/AA–SFR1, SWI5^F/A–SFR1 and SWI5^L/A–SFR1 mutants were examined for RAD51 interaction by affinity pulldown. (E) The results from (D) were graphed. Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.

Figure 4.

Biophysical properties of SWI5 F83A/L85A-SFR1 mutant complex. (A) Analytical ultracentrifugation with sedimentation velocity analysis of SWI5–SFR1 and SWI5 F83A/L85A–SFR1 complexes. The experimental data were analyzed by the Sedfit program (version 14.1), which yielded an estimated molecular mass of 45.8 kDa for SWI5–SFR1, and 44.7 kDa for SWI5 F83A/L85A–SFR1. (B) Circular dichroism spectra of SWI5–SFR1 and SWI5 F83A/L85A–SFR1 complexes. The spectra showed no significant difference in secondary structure between SWI5–SFR1 and SWI5 F83A/L85A–SFR1.

Figure 5.

SWI5 F83A/L85A-SFR1 is functionally impaired. (A) The effect of SWI5–SFR1 or SWI5^FL/AA–SFR1 on RAD51-mediated DNA strand exchange was examined. The results were graphed. (B) Exonuclease I protection assay was conducted with the indicated concentrations of SWI5–SFR1 and SWI5^FL/AA–SFR1. The results were graphed. (C) The average length of RAD51 filaments with negative staining was determined by electron microscopy (see Supplementary Figure S5). RAD51 was examined alone or with SWI5–SFR1 or SWI5^FL/AA–SFR1. The total 225 filaments were counted in each reaction. We note that the average length of the presynaptic filament in the presence of SWI5–SFR1 is much longer than expected. This could be due to the end-to-end association between two DNA molecules by RAD51 as described by Baumann et al. (35). (D) Thin-layer chromatography to monitor the hydrolysis of [γ-³²P] ATP by RAD51 in the absence or presence of indicated concentrations of SWI5–SFR1 or SWI5^FL/AA–SFR1. The results were graphed. (A, B and D) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.