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.

RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination.

The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.

Tryptophan biosynthesis is important for resistance to replicative stress in Saccharomyces cerevisiae.

Acute tryptophan depletion is used to induce low levels of serotonin in the brain. This method has been widely used in psychiatric studies to evaluate the effect of low levels of serotonin, and is generally considered a safe and reversible procedure. Here we use the budding yeast Saccharomyces cerevisiae to study the effects of tryptophan depletion on growth rate upon exposure to DNA-damaging agents. Surprisingly, we found that budding yeast undergoing tryptophan depletion were more sensitive to DNA-damaging agents such as methyl methanesulphonate (MMS) and hydroxyurea (HU). We found that this defect was independent of several DNA repair pathways, such as homologous recombination, base excision repair and translesion synthesis, and that this damage sensitivity was not due to impaired S-phase signalling. Upon further analysis, we found that the DNA-damage sensitivity of tryptophan depletion was likely due to impaired protein synthesis. These studies describe an important source of variance in budding yeast when using tryptophan as an auxotrophic marker, particularly on studies focusing on DNA repair, and suggest that further testing of the effect of tryptophan depletion on DNA repair in mammalian cells is warranted. Copyright © 2016 John Wiley & Sons, Ltd.