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. 2026 Oct 3;17:11324. doi: 10.1038/s41467-026-77941-y

Fig. 6. RAD51D HR deficient variants differentially impact ssDNA binding, BCDX2 ATPase activity, and/or RAD51-mediated strand exchange.

Fig. 6

a Coomassie-stained SDS–PAGE gel analysis of the purified CX3, BC, and DX2 sub-complexes carrying either WT or RAD51D variants, one representative gel shown. b Pull-down analysis of WT BC (RAD51B-His/RAD51C) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. Mean values ± s.d. from two independent are indicated below. c ssDNA binding of BCDX2 paralog complexes reconstituted by mixing WT BC and DX2 sub-complexes with indicated RAD51D variants. Results from three independent experiments were plotted as mean values ± s.d. The p-values for the significance of differences were calculated using two-way ANOVA with DX2 variants and Protein concentration as fixed factors, followed by Dunnett’s post-hoc test for multiple comparisons against the Wild-Type (WT) group. d ATPase analysis of 0.5 μM WT BC and DX2 sub-complexes with indicated RAD51D variants in the presence of ssDNA after 60 min incubation. Pi indicates released inorganic phosphate after hydrolysis. The histograms represent mean values ± s.d. of three independent experiments. Statistical significance between mean values was assessed using an unpaired two-sided t-test. e Pull-down analysis of WT CX3 (RAD51C-His/XRCC3-STREP) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. Mean values ± s.d. of the pulled-down from two independent experiments are indicated below. f ssDNA binding of X3CDX2 paralog complexes reconstituted by mixing WT CX3 and DX2 sub-complexes with indicated RAD51D variants. Results from three independent experiments were plotted as mean values ± s.d. The p-values were calculated using two-way ANOVA with DX2 variants and protein concentration as fixed factors, followed by Dunnett’s post-hoc test for multiple comparisons against the Wild-Type (WT) group. g Reaction schematic of DNA strand exchange in the presence of RPA. (h) DNA strand exchange in the absence or presence of 30 nM RPA, with 100 nM RAD51 alone or a mixture of RAD51 and indicated RAD51 paralog complexes. Reactions were performed with AMP-PNP as the nucleotide cofactor. The positions of the Cy5‑labeled (indicated by asterisk) 40‑bp dsDNA and the strand‑exchange product are annotated alongside the gel images. Mean values from four independent experiments are plotted ± s.d. Statistical significance between mean values was assessed using an unpaired two-sided t-test. Source data are provided as a Source Data file.