Abstract
The tumor suppressor RAD51D is essential for homologous recombination (HR). Pathogenic variants in RAD51D are associated with breast and ovarian cancers. However, most clinical missense variants are of uncertain significance. Here, we report a multiplex assay to test 6,888 RAD51D coding variants for loss-of-function. The resulting variant-to-function map perfectly separates known pathogenic and benign variants and is validated by orthogonal HR and biochemical assays across 70 clinical variants. Our screen shows that variants in the DNA-binding or ATPase core most severely compromise HR, and we identify the RAD51D-RAD51C interface within the BCDX2 complex as essential for regulating its ATPase activity. We hypothesize that, paradoxically, RAD51D’s primary function is to slow BCDX2 ATPase activity, thereby allowing sufficient time and space for RAD51 filament assembly. Together, we identify hotspots of deleterious RAD51D variants and uncover how variants compromise its biochemical functions.