BRCA1 Mutation and Repair Assay

MMC (SCBT, Santa Cruz, CA, USA), cisplatin (Sigma-Aldrich, St Louis, MO, USA), ABT-888 (SelleckChem, Houston, TX, USA) and bleomycin (SCBT) were purchased in dimethyl sulfoxide or prepared as stocks in dimethyl sulfoxide. The Brca1 deletion mutants^{36 (link)} are HA tagged and correspond to deletions within the Brca1 protein as illustrated in Figure 6d. The Brca1 ΔM3 mutant (amino acids 1290–1530) was generated from full-length HA-Brca1 plasmid³⁷ using methods described in Gibson Assembly Cloning Kit (New England BioLabs, Ipswich, MA, USA) and primer sequences: BRCA1-305-For, 5′-CAGAATGAATGTAGAAAAGGCTG-3′ and BRCA1-305-1290-del-Rev, 5′-GTTGCTCCTCCACATCAACAACACATTTTGTTTCCTCACTAAG-3′; BRCA1-305-1290-del-For, 5′-CTTAGTGAGGAAACAAAATGTGTTGTTGATGTGGAGGAGCAAC-3′ and pcDNA-Xho-rev, 5′-TAGGGCCCTCTAGATGCATGC-3′. XhoI and EcoRI restriction digestion and sequencing validated the correct insertion and sequence. The full-length myc-Wwox plasmid^{13 (link)} and deletion mutants^{15 (link)} were described previously.