Fig. 1. Mouse ES cell-based functional assay for BRCA2 variants.

a Schematic representation of the functional assay. BAC DNA encoding human BRCA2 gene with any variant was introduced into PL2F7 mES cells containing a conditional allele and a knockout allele of Brca2. Conditional allele was further deleted by CRE and the recombinants were selected on HAT containing media. Depending on the impact of BRCA2 variants, HAT^r cells may or may not be viable. Viable HAT^r cells were further tested for sensitivity to different DNA damaging agents to distinguish between variants that have no impact on function and those that have some loss of function (hypomorphic). Star in the BAC construct represents variant. Two halves of HPRT mini gene are marked in solid boxes as HP and RT. Solid arrows denote loxP sites. b Schematic diagram of BRCA2 protein with different domains and position of variants selected. Different domains are marked as colored boxes and the amino acids (aa) for the respective domains are noted below. HD helical domain, DBD DNA-binding domain, OB oligonucleotide binding fold, TR2 C-terminal RAD51-binding site, NLS nuclear localization signal^15,43,44. Variants selected for analysis are denoted as colored solid circles on top. Missense, silent or synonymous and nonsense variants are marked in red, blue, and black colors, respectively.