Yeast Two-Hybrid Plasmid Generation

To generate the yeast two-hybrid plasmids, full-length (1–1143) or C-term (620–1143) was inserted into pGBKT7 (Clontech) using SmaI/SalI restrictions sites. The BRCv (687FR→AA) mutation was created by Quikchange mutagenesis (Agilent Genomics) using Kapa DNA polymerase (Kapa Biosystems) and screened with novel silent restriction sites. RAD51 was PCR amplified from pGAT3-RAD51 (81 (link)) with primers that included MfeI and XhoI sites and then ligated into pGADT7 (Clontech) digested with EcoRI/XhoI. The yeast strain SFY526 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, canr, gal4-542, gal80–538, URA::GAL1UAS-GAL1TATA-lacZ) was transformed with the appropriate plasmids (see figure legends) according to the manufacturer's instructions using the lithium acetate procedure (Clontech Matchmaker 2 manual). Liquid cultures were grown overnight in standard dropout (SD) media lacking tryptophan and leucine. The cells were restreaked onto SD/-Trp/-Leu and incubated at 30 °C for 2 to 3 days before performing a colony-lift β-galactosidase assay (X-gal, Sigma-Aldrich) according to the Yeast Protocols Handbook (PT3024-1, Clontech).