Fig. 1

Functional analysis of BRCA2 variants in Brca2^cko/ko mES cells.a Schematic representation of the mES cell-based functional assay. b Expression of BRCA2 variants in mES cells by Western blotting. Two independent clones were generated for each variant. Vinculin was used as loading control. c Expression of BRCA2 P3039P by RT-PCR using primers from exons 20 (5′-AGGAAGAAAAGGAAGCAGCAAAATATGTGG-3′) and 25 (5′-TCTCCAGCAAATAAAGTAAGAAGG-3′) revealed lack of full-length transcript. Two alternatively spliced transcripts were observed. d Sequence analysis of the two transcripts revealed major alternatively spliced transcript skipped exon 23 (lower band) and a minor form that skipped exon 23 but retained 63pb of intron 22 (upper band). e Quantification of viability of Brca2^ko/ko mES cell by various BRCA2 variants. Two independent BAC clones expressing the variants were analyzed. Average numbers of viable cells from two independent clones were plotted. mES cell clone expressing WT BRCA2 was used as control. f Clonogenic survival assay confirming sensitivity of mES cells expressing A262T to olaparib (P value < 0.001 at 100 nM and < 0.0001 at 500 nM concentrations using a multiple t-test). g Representative images showing γH2AX and RAD51 foci at 3, 6, and 10 h post 10 Gy IR in mES cells expressing WT and A262T BRCA2. h Quantification of γH2AX and RAD51 foci at 3, 6, and 10 h post 10 Gy IR in mES cells expressing WT and A262T BRCA2. i Quantification of homologous recombination using a GFP-based HR reporter in mES cells expressing WT and A262T BRCA2 (P values are 0.008 for Clone 1 and 0.01 for Clone 2 using paired t-test)