Fig. 5. A subset of RAD51D breast and ovarian cancer identified variants alter interaction with XRCC2 or RAD51C by Y2H or Y3H, respectively.

a, b Interaction proficiency of RAD51D VUS was measured via (a) yeast 2-hybrid (Y2H) of pGAD-RAD51D or its variant, expressed in a GAL4 DNA activating domain expressing plasmid with pGBD-XRCC2 expressed in the GAL4 DNA binding domain expressing plasmid and (b) pGAD-RAD51D or its variant, expressed in the pGAD (GAL4 DNA activating domain) plasmid with pGBD-RAD51C (GAL4 DNA binding domain) plasmid with pADH1-RAD51B via yeast 3-hybrid (Y3H). RAD51B serves to stabilize RAD51C protein levels. Empty vectors are used as a negative control. Quantification of yeast growth from three experiments is plotted as mean ± s.d relative to the WT control. Representative images for variants and controls are shown. Variants with <50% interaction are classified as deficient and plotted in red. c RAD51D variants with reduced interaction with XRCC2 or RAD51C are expressed. Western blot analysis of protein extract from yeast cells in (a) expressing WT RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed by measuring the amount of Kar2 using an anti-Kar2 antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate. See Supplementary Fig. 7 for Western blots of all variants. d–f MAVE function scores vs individual variant assay results of (d) HR activity, p-value determined by simple linear regression, (e) XRCC2 binding, and (f) RAD51C binding. Missense variant point shape denotes functional status (LOF/INT/NEU) based upon MAVE result. Source data are provided as a Source Data file.