Homologous recombination-deficient mutation cluster in tumor suppressor RAD51C identified by comprehensive analysis of cancer variants.

Mutations in homologous recombination (HR) genes, including BRCA1, BRCA2, and the RAD51 paralog RAD51C, predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

Variants in the first methionine of RAD51C are homologous recombination proficient due to an alternative start site.

In the 20+ years since the discovery of RAD51C, scientists have been perplexed as to how missense variants in this tumor suppressor gene impacts its function and pathogenicity. With a strong connection to breast and ovarian cancer, classifying these variants as pathogenic or benign aids in the diagnosis and treatment of patients with RAD51C variants. In particular, variants at translational starts sites are disruptive as they prevent protein expression. These variants are often classified as pathogenic, unless an alternative translational start is shown to produce a functional isoform to rescue protein expression. In this study, we utilized the ribosome profiling database GWIPS-VIZ to identify two active translational start sites in human RAD51C at methionine one and methionine ten. This second translational start at methionine ten is both conserved in 97 % of mammals and is the sole translational start in 80 % of mammals. Missense variants at either methionine have been identified in 47 individuals, preventing expression from one of these two start sites. Therefore, we stably expressed both wildtype isoforms, as well as the RAD51C M1 and M10 variants in a RAD51C CRISPR/Cas9 knockout U2OS cell and compared their homologous recombination function. Surprisingly, we find that expression of human RAD51C from either start site can equivalently rescue homologous recombination of RAD51C CRISPR/Cas9 knockout U2OS cells through a sister chromatid recombination assay. Similarly, each of our RAD51C CRISPR/Cas9 KO cells stably complemented with RAD51C missense variants at either M1 or M10 are homologous recombination proficient. Together, our data demonstrate that RAD51C has two translational start sites and that variants in either methionine result in homologous recombination proficiency. With this critical discovery, individuals with variants at M1 will be more accurately informed of their cancer risk upon reclassification of these variants.

The human Shu complex promotes RAD51 activity by modulating RPA dynamics on ssDNA.

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1 and SWS1. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter SWS1 complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.

Finding significance: New perspectives in variant classification of the RAD51 regulators, BRCA2 and beyond.

For many individuals harboring a variant of uncertain functional significance (VUS) in a homologous recombination (HR) gene, their risk of developing breast and ovarian cancer is unknown. Integral to the process of HR are BRCA1 and regulators of the central HR protein, RAD51, including BRCA2, PALB2, RAD51C and RAD51D. Due to advancements in sequencing technology and the continued expansion of cancer screening panels, the number of VUS identified in these genes has risen significantly. Standard practices for variant classification utilize different types of predictive, population, phenotypic, allelic and functional evidence. While variant analysis is improving, there remains a struggle to keep up with demand. Understanding the effects of an HR variant can aid in preventative care and is critical for developing an effective cancer treatment plan. In this review, we discuss current perspectives in the classification of variants in the breast and ovarian cancer genes BRCA1, BRCA2, PALB2, RAD51C and RAD51D.

RAD51 paralog function in replicative DNA damage and tolerance.

RAD51 paralog gene mutations are observed in both hereditary breast and ovarian cancers. Classically, defects in RAD51 paralog function are associated with homologous recombination (HR) deficiency and increased genomic instability. Several recent investigative advances have enabled characterization of non-canonical RAD51 paralog function during DNA replication. Here we discuss the role of the RAD51 paralogs and their associated complexes in integrating a robust response to DNA replication stress. We highlight recent discoveries suggesting that the RAD51 paralogs complexes mediate lesion-specific tolerance of replicative stress following exposure to alkylating agents and the requirement for the Shu complex in fork restart upon fork stalling by dNTP depletion. In addition, we describe the role of the BCDX2 complex in restraining and promoting fork remodeling in response to fluctuating dNTP pools. Finally, we highlight recent work demonstrating a requirement for RAD51C in recognizing and tolerating methyl-adducts. In each scenario, RAD51 paralog complexes play a central role in lesion recognition and bypass in a replicative context. Future studies will determine how these critical functions for RAD51 paralog complexes contribute to tumorigenesis.

Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex.

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase ß and a de novo DNA Methyltransferase.

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from -189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase ß (pol ß). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol ß and the de novo DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and de novo DNA methylation.

Modulation of trinucleotide repeat instability by DNA polymerase ß polymorphic variant R137Q.

Trinucleotide repeat (TNR) instability is associated with human neurodegenerative diseases and cancer. Recent studies have pointed out that DNA base excision repair (BER) mediated by DNA polymerase ß (pol ß) plays a crucial role in governing somatic TNR instability in a damage-location dependent manner. It has been shown that the activities and function of BER enzymes and cofactors can be modulated by their polymorphic variations. This could alter the function of BER in regulating TNR instability. However, the roles of BER polymorphism in modulating TNR instability remain to be elucidated. A previous study has shown that a pol ß polymorphic variant, polßR137Q is associated with cancer due to its impaired polymerase activity and its deficiency in interacting with a BER cofactor, proliferating cell nuclear antigen (PCNA). In this study, we have studied the effect of the pol ßR137Q variant on TNR instability. We showed that pol ßR137Q exhibited weak DNA synthesis activity to cause TNR deletion during BER. We demonstrated that similar to wild-type pol ß, the weak DNA synthesis activity of pol ßR137Q allowed it to skip over a small loop formed on the template strand, thereby facilitating TNR deletion during BER. Our results further suggest that carriers with pol ßR137Q polymorphic variant may not exhibit an elevated risk of developing human diseases that are associated with TNR instability.