Rawal, Y. | Search bioRxiv

bioRxiv2026

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

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. HighlightsO_LIUsed 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 C_LIO_LIProvided cellular functional analysis for 70 clinically-identified breast and ovarian cancer RAD51D variants C_LIO_LIIdentified key regions and enzymatic activities of RAD51D critical for its function in the BCDX2 and the X3CDX2 complexes C_LIO_LIDetermined mechanism of RAD51D-mediated regulation of BCDX2 ATPase activity C_LI

cancer biology↗

bioRxiv2022

Distinct functions of three chromatin remodelers in activator binding and preinitiation complex assembly

The nucleosome remodeling complexes (CRs) SWI/SNF, RSC, and Ino80C cooperate in evicting or repositioning nucleosomes to produce nucleosome depleted regions (NDRs) at the promoters of many yeast genes induced by amino acid starvation. We analyzed mutants depleted of the catalytic subunits of these CRs for binding of transcriptional activator Gcn4 and recruitment of TATA-binding protein (TBP) during preinitiation complex (PIC) assembly. RSC and Ino80 were found to enhance Gcn4 binding to both UAS elements in NDRs upstream of promoters and to unconventional binding sites within nucleosome- occupied coding sequences; and SWI/SNF contributes to UAS binding when RSC is depleted. All three CRs are actively recruited by Gcn4 to most UAS elements and appear to enhance Gcn4 binding by reducing nucleosome occupancies at the binding motifs, indicating a positive regulatory loop. SWI/SNF acts unexpectedly in WT cells to prevent excessive Gcn4 binding at many UAS elements, indicating a dual mode of action that is modulated by the presence of RSC. RSC and SWI/SNF collaborate to enhance TBP recruitment at Gcn4 target genes, together with Ino80C, in a manner associated with nucleosome eviction at the TBP binding sites. Cooperation among the CRs in TBP recruitment is also evident at the highly transcribed ribosomal protein genes, while RSC and Ino80C act more broadly than SWI/SNF at the majority of other constitutively expressed genes to stimulate this step in PIC assembly. Our findings indicate a complex interplay among the CRs in evicting promoter nucleosomes to regulate activator binding and stimulate PIC assembly. AUTHOR SUMMARYATP-dependent chromatin remodelers (CRs), including SWI/SNF and RSC in budding yeast, are thought to stimulate transcription by repositioning or evicting promoter nucleosomes, and we recently implicated the CR Ino80C in this process as well. The relative importance of these CRs in stimulating activator binding and recruitment of TATA-binding protein (TBP) to promoters is incompletely understood. Examining mutants depleted of the catalytic subunits of these CRs, we determined that RSC and Ino80C stimulate binding of transcription factor Gcn4 to nucleosome-depleted regions, or linkers between genic nucleosomes, at multiple target genes activated by Gcn4 in amino acid-starved cells, frequently via evicting nucleosomes from the Gcn4 binding motifs. At some genes, SWI/SNF functionally complements RSC, while opposing RSC at others to limit Gcn4 binding. The CRs in turn are recruited by Gcn4 to establish positive or negative feedback loops that control Gcn4 binding. The three CRs also cooperate to enhance TBP recruitment, again involving nucleosome depletion, at both Gcn4 target and highly expressed ribosomal protein genes, whereas only RSC and Ino80C act broadly throughout the genome to enhance this key step in preinitiation complex assembly. Our findings illuminate functional cooperation among multiple CRs in regulating activator binding and promoter activation.

molecular biology↗