RAD51C is a tumor suppressor important during the homologous recombination (HR) DNA repair pathway [1]. HR is a high-fidelity double-strand break repair mechanism that uses the homologous template of the sister chromatid or homologous chromosome to repair damaged DNA [2]. Defects in HR are highly associated with an increased risk of breast and ovarian cancer and HR deficient cancers can be targeted through the use of precision therapeutics like PARP inhibitors [3]. RAD51C comes from a family of proteins that derive from a gene duplication event of the ATPase RAD51 and is therefore a RAD51 paralog [2]. RAD51C is unique in that it interacts with the other RAD51 paralogs to form two distinct complexes the BCDX2 complex with RAD51B, RAD51D, and XRCC2 and the CX3 complex with XRCC3 [2]. While RAD51C is the most well studied of the RAD51 paralogs, RAD51D has also been established as a tumor suppressor and its disruption has connections to both breast and ovarian cancer [1]. Like RAD51D, variants in RAD51C have been identified in several hereditary breast and ovarian cancer (HBOC) families [4], [5], [6], [7]. Yet, the majority of these variants are classified as variants of unknown significance (VUS) and therefore cannot aid in risk assessment or expanding treatment options (ClinVar).

RAD51C variants currently classified as pathogenic are generally those which greatly alter protein structure and expression. This includes early stop codon and large deletions. However, variants in RAD51C’s ATG start codon are still not well understood. Confounding this issue, germline variants in the first methionine of RAD51C have been identified in 21 different individuals in ClinVar. Suggesting that there may be an alternative start site, a second methionine in RAD51C is located in frame at amino acid 10. In 2018, the ClinGen Sequence Variant Interpretation Working Group (SVI) released their updated criteria for interpretation of loss-of-function variant criteria [8]. This new guidance expands on the loss of protein expression criteria (pathogenic very strong [PVS1]) and notes that functional isoforms produced from an alternative translational start must be considered before use of this evidence criteria [8]. Therefore, without knowledge of RAD51C’s translational start, we cannot determine how variants in RAD51C’s translational start affect protein expression, RAD51C function or cancer risk.

Here we asked whether RAD51C has two translational start sites and how germline variants in methionine one or ten impact RAD51C HR function. We found that 97% of mammalian species are conserved with human RAD51C (hRAD51C) at the M10 position. Importantly, this second methionine is the sole start site in 80% of mammals with complete RAD51C sequences. By creating RAD51C isoforms that translate RAD51C exclusively at M1 or M10 positions, we find that both RAD51C isoforms are HR proficient. Ribosome profiling reveals that, in multiple human cell lines, translation can initiate from two distinct ATG sequences. Consistent with this, we find that patient derived variants in M1 and M10 are HR proficient (ClinVar and gnomAD). Our results demonstrate that, in vivo, hRAD15C has two fully functional translational start sites and that protein translation from either start maintains HR proficiency. These findings have important implications for determining cancer risk and treatment strategies for patients with RAD51C variants in M1 or M10.