Fig. 4. Breast and ovarian cancer-identified variants in the ATPase and DNA-binding regions of RAD51D exhibit reduced HR proficiency.

a HR proficiency of 70 RAD51D missense variants of unknown significance. HR function was classified in comparison to the range of synonymous (green, HR > 0.75) and truncation variants (red, HR < 0.6). Missense variants are colored corresponding to function (HR > 0.7 in grey; intermediate yellow, and HR < 0.6 in light red). See Supplementary Fig. 5b, c for all synonymous and truncation variants tested, and note that a subset of those variants analyzed are replotted here as representative variants. The experiment was performed independently 4–9 times per variant (see Source Data for exact n of each variant) and plotted as mean values ± s.d. b, e Olaparib and cisplatin sensitivity of breast/ovarian cancer identified RAD51D variants with reduced HR. Representative images of U2OS RAD51D KO cell lines stably expressing pcDNA3 RAD51D WT or the indicated RAD51D variant that were treated with increasing concentrations of Olaparib (b) or cisplatin (d). c, e Clonogenic survival assays were quantified by percent colony area and normalized to the area of untreated or vehicle control. Means of 4–12 trials (see Source Data for precise n for each variant) are plotted ± s.d., and drug concentrations with colony area <0.001 are omitted. f RAD51D variants with reduced HR are expressed. Western blot analysis of U2OS RAD51D KO cell lines stably expressing WT or the indicated RAD51D variants. RAD51D protein expression was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-Tubulin antibody. Note that protein expression can be variable, but notably L4H consistently exhibits reduced protein expression. Experiment performed in triplicate. g Structures of the RAD51 paralog pentamer X3CDX2 (PDB: 9SVX) and BCDX2 (PDB: 8GBJ) complexes. Cartoon representations of XRCC2 (purple), RAD51D (light blue), RAD51C (green), XRCC3 (yellow), RAD51 (orange), RAD51B (red), bound ATP (yellow-orange), and ssDNA (orange) are shown consistently throughout. h, i Variants causing complete (red, h) or intermediate (yellow, i) deficiency in homologous recombination are highlighted on RAD51D. The interacting surfaces of RAD51C and XRCC2 are outlined with black dashed lines. The contact interfaces are identical between the XRCC3 and BCDX2 complex. j, k Variants that disrupt RAD51C-RAD51D interactions are shown as red sticks, mapped onto the BCDX2 complex structure. Source data are provided as a Source Data file.