Pmh sfb brca1

A pCDH-BRCA1 plasmid for the mammalian expression of FL BRCA1 was generated by subcloning FL BRCA1 cDNA from pMH-SFB-BRCA1 (Addgene). Plasmids expressing FLAG-tagged WT or S1655A mutant BRCA1 BRCT domains were generated by replacing FL BRCA1 with the corresponding cDNAs along with a nuclear localization signal in pMH-SFB-BRCA1. To generate a GST-tagged BRCT fragment, the corresponding coding sequence was PCR amplified from pMH-SFB-BRCA1 and cloned into pGEX-4T1 (GE Healthcare) between the EcoRI and SalI sites. The mutagenesis primer 5′-GAA TGT CCA TGG TGG TGG CTG GCC TGA CCC CAG AA -3′ was used to generate pGEX-4T1-S1655A. To induce ectopic expression of as-5.8S rRNA, the corresponding as-5.8S rRNA sequence was amplified by PCR using a 5′-AAG CGA CGC TCA GAC AGG CG-3′ forward primer and a 5′-CGA CTC TTA GCG GTG GAT CA-3′ reverse primer, and the PCR product was cloned into an inducible lentiviral pLKO vector (Addgene). as-5.8S mutant rRNA sequence was generated using pLKO-as-5.8S as the template with the following mutagenesis primers: 5′- AGA CAG GCG TAG CCC CGG GCC GCA AGT GCG TT-3′, 5′-CGG GCC GCA AGT GCG TTG TGT GTC CTG CAA TTC ACA TT-3′, 5′-TCC TGC AAT TCA CAT TGG GCC GCA AGT GCG TT -3′, 5′-TGG GCC GCA AGT GCG TTC GAG TGA TCC ACC GCT A-3′. Plasmid 1GFP/RNase H1 D210N for expressing GFP-His₆-RNase H1 D210N in E. coli was purchased from Addgene.