Enhancement of ADP release from the RAD51 presynaptic filament by the SWI5-SFR1 complex

Abstract

Homologous recombination catalyzed by the RAD51 recombinase eliminates deleterious DNA lesions from the genome. In the presence of ATP, RAD51 forms a nucleoprotein filament on single-stranded DNA, termed the presynaptic filament, to initiate homologous recombination-mediated DNA double-strand break repair. The SWI5-SFR1 complex stabilizes the presynaptic filament and enhances its ability to mediate the homologous DNA pairing reaction. Here we characterize the RAD51 presynaptic filament stabilization function of the SWI5-SFR1 complex using optical tweezers. Biochemical experiments reveal that SWI5-SFR1 enhances ATP hydrolysis by single-stranded DNA-bound RAD51. Importantly, we show that SWI5-SFR1 acts by facilitating the release of ADP from the presynaptic filament. Our results thus provide mechanistic understanding of the function of SWI5-SFR1 in RAD51-mediated DNA recombination.

Figure 1.

SWI5-SFR1 stabilizes RAD51 filament. (A) Schematic of the optical tweezers setup to monitor the presynaptic filament length. (B) Histograms of contour lengths of individual naked 6123-bp duplex DNA molecules alone, in the presence of RAD51 and ATP, or the mixture of RAD51, SWI5-SFR1 and ATP.

Figure 2.

SWI5-SFR1 enhances RAD51 ATPase activity. (A) Thin layer chromatography to monitor the hydrolysis of [γ-³²P] ATP by RAD51. The asterisk denotes the ³²P label. Ca²⁺ inhibits ATP hydrolysis, whereas SWI5-SFR1 stimulates hydrolysis in a concentration-dependent (B) and time-dependent (C) manner. (D) The stimulatory effect of ATP hydrolysis by SWI5-SFR1 is dependent on ssDNA. (B–D) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, SWI5-SFR1.

Figure 3.

Functional interactions between RAD51 and SWI5-SFR1 complex. (A) SWI5-SFR1 complex but not SWI5 or SFR1 enhances ATP hydrolysis by the RAD51 presynaptic filament. (B) No physical interaction was seen between mouse SWI5-SFR1 and S. cerevisiae Rad51 (ScRad51) by affinity pulldown. The supernatant (S), wash (W) and SDS eluate (E) from the pulldown reaction were analyzed by SDS–PAGE. (C) SWI5-SFR1 has no effect on ATP hydrolysis by the ScRad51 presynaptic filament. (D) SWI5-SFR1 has no effect on ScRad51-mediated homologous DNA pairing. The results were graphed. The asterisk denotes the ³²P label in the DNA strand. (E) RAD51AP1 has no effect on ATP hydrolysis by the RAD51 presynaptic filament. (A, C, D and E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbols: S5S1, SWI5-SFR1; ScRad51, S. cerevisiae Rad51.

Figure 4.

SWI5-SFR1 but not RAD51AP1 mediates ADP–ATP exchange of RAD51 filament. (A) Schematic of the filter-binding assay to monitor ADP–ATP exchange within the RAD51-ssDNA presynaptic filament. (B) SWI5-SFR1 facilitates ADP–ATP exchange of RAD51 filament in a dosage-dependent manner. (C) There is no enhancement of nucleotide exchange by SWI5-SFR1 on the omission of ssDNA. (D) RAD51AP1 lacks the ability to facilitate ADP–ATP exchange within the RAD51 presynaptic filament. (B–D) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, SWI5-SFR1.

Figure 5.

SWI5-SFR1 expedites ADP release from the RAD51 presynaptic filament. (A) Schematic of the filter-binding assay to monitor ADP release from the RAD51 presynaptic filament. (B) SWI5-SFR1 facilitates ADP release from RAD51 presynaptic filament in a protein concentration- and ssDNA-dependent manner. (C) RAD51AP1 lacks the ability to facilitate ADP release from the presynaptic filament. (B and C) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, SWI5-SFR1.

Figure 6.

SWI5-SFR1 does not alter ATP-binding affinity of RAD51 presynaptic filament. (A) Schematic of the filter-binding assay to monitor the influence of SWI5-SFR1 on the nucleotide-binding affinity of RAD51 in the presence of Ca²⁺ ions. (B) SWI5-SFR1 has no significant effect on ATP binding by RAD51. (C) Even with Ca²⁺ present, attenuation of the ADP-binding affinity of RAD51 presynaptic filament by SWI5-SFR1 still occurred. (B and C) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, SWI5-SFR1.

Figure 7.

Model depicting the mechanistic action of SWI5-SFR1 on RAD51 filament. Our results show that SWI5-SFR1 helps maintain the catalytically active ATP-bound state of the RAD51 presynaptic filament via enhancement of ADP release from the filament.