High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

Summary 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 unknown significance. We performed a multiplex assay of variant effect 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, the primary function of RAD51D is to slow the ATPase activity of BCDX2, thereby allowing sufficient time and space for RAD51 filament assembly. Together, we identify hotspots of deleterious RAD51D variants and uncover the mechanisms by which variants compromise its biochemical functions. Highlights Used a multiplexed assay of functional effect (MAVE) to assess the functionality via olaparib sensitivity of 6,888 RAD51D coding variants, which can be used for variant classification Provided cellular functional analysis for 70 clinically-identified breast and ovarian cancer RAD51D variants Identified key regions and enzymatic activities of RAD51D critical for its function in the BCDX2 and the X3CDX2 complexes Determined mechanism of RAD51D-mediated regulation of BCDX2 ATPase activity