Journal: Nature

Article Title: Promotion of RAD51-mediated homologous DNA pairing by BRCA1-BARD1

doi: 10.1038/nature24060

Figure Lengend Snippet: a, Binding of D-loop, DNA bubble (Bubble), replication fork (RF), dsDNA and ssDNA. b, Quantification of a. Data are means ± s.d., n=3 or 5. c, Southwestern analysis to test Dloop binding. BSA was the negative control. d, Pulldown analysis for interaction of RAD51 or yRad51 with BRCA1-BARD1. e, Far Western analysis for interaction of BRCA1 and BARD1 with RAD51. B1-B1, BRCA1-BARD1. BSA and RAD54 were the negative and positive controls, respectively.

Article Snippet: After centrifugation (100,000 × g for 90 min), the clarified lysate was incubated with 2 ml Glutathione Sepharose 4 Fast Flow resin (GE Healthcare; for GST-BARD1 123-162 ) or Ni-NTA resin (GE healthcare; for BARD1 124-270 ) for 2 h. The affinity resin was transferred to a glass column (1.5 × 15 cm), washed with 20 ml buffer D, before being eluted 3 times with 3 ml of 20 mM glutathione or 150 mM imidazole in buffer D. For BARD1 124-270 , the His 6 -SUMO tag was cleaved by the Ulp1 protease by an overnight incubation at 4°C.

Techniques: Binding Assay, Negative Control, Western Blot

Journal: Nature

Article Title: Promotion of RAD51-mediated homologous DNA pairing by BRCA1-BARD1

doi: 10.1038/nature24060

Figure Lengend Snippet: a, Assay schematic. b, Reactions with BRCA1-BARD1 and BRCA2-DSS1. c, Quantification of b. Data are means ± s.d., n=3. d, Cartoon depicting the role of BRCA1-BARD1 in DNA strand invasion.

Article Snippet: After centrifugation (100,000 × g for 90 min), the clarified lysate was incubated with 2 ml Glutathione Sepharose 4 Fast Flow resin (GE Healthcare; for GST-BARD1 123-162 ) or Ni-NTA resin (GE healthcare; for BARD1 124-270 ) for 2 h. The affinity resin was transferred to a glass column (1.5 × 15 cm), washed with 20 ml buffer D, before being eluted 3 times with 3 ml of 20 mM glutathione or 150 mM imidazole in buffer D. For BARD1 124-270 , the His 6 -SUMO tag was cleaved by the Ulp1 protease by an overnight incubation at 4°C.

Techniques:

Journal: Nature

Article Title: Promotion of RAD51-mediated homologous DNA pairing by BRCA1-BARD1

doi: 10.1038/nature24060

Figure Lengend Snippet: a, Synaptic complex assay schematic. b, Synaptic complex formation by the RAD51-ssDNA filament and BRCA1-BARD1. c, Quantification of b. Data are means ± s.d., n=3 or 6. d, DNA curtain assay schematic39,40. e, Number of dsDNA oligonucleotides bound by each RAD51-ssDNA or yRad51-ssDNA filament as a function of BRCA1-BARD1 concentration. Data are means ± 95% confidence intervals, n=49, 50, 38, 54, 51 or 53. f, Binding distribution for Atto565-dsDNA with or without BRCA1-BARD1. g, Semi-log survival plot of the synaptic complex with and without 100 nM BRCA1-BARD1. *, P<0.05; **, P<0.01. NS=non-significant. In f and g, data are means ± errors (determined by bootstrapping). The multiguassian in f and the lines in g were fitted with least squares analysis.

Article Snippet: After centrifugation (100,000 × g for 90 min), the clarified lysate was incubated with 2 ml Glutathione Sepharose 4 Fast Flow resin (GE Healthcare; for GST-BARD1 123-162 ) or Ni-NTA resin (GE healthcare; for BARD1 124-270 ) for 2 h. The affinity resin was transferred to a glass column (1.5 × 15 cm), washed with 20 ml buffer D, before being eluted 3 times with 3 ml of 20 mM glutathione or 150 mM imidazole in buffer D. For BARD1 124-270 , the His 6 -SUMO tag was cleaved by the Ulp1 protease by an overnight incubation at 4°C.

Techniques: Concentration Assay, Binding Assay

Journal: Nature

Article Title: Promotion of RAD51-mediated homologous DNA pairing by BRCA1-BARD1

doi: 10.1038/nature24060

Figure Lengend Snippet: a, Domains in BRCA1-BARD1. b, Alignment of the RAD51 interaction domain in BARD1 orthologs. The highlighted residues (in green) were changed to AAE or N (in red). The asterisks denote BARD1 mutations found in human cancers (cBioPortal for Cancer Genomic). c, Testing of RAD51 interaction with wild type or mutant BRCA1-BARD1. d, Examination of BRCA1-BARD1 mutants in the D-loop reaction. e, Quantification of d. Data are means ± s.d., n=3, 4 or 5. P values were calculated using two-way ANOVA and multiple comparisons were corrected by the Bonferroni method. **, P<0.01.

Article Snippet: After centrifugation (100,000 × g for 90 min), the clarified lysate was incubated with 2 ml Glutathione Sepharose 4 Fast Flow resin (GE Healthcare; for GST-BARD1 123-162 ) or Ni-NTA resin (GE healthcare; for BARD1 124-270 ) for 2 h. The affinity resin was transferred to a glass column (1.5 × 15 cm), washed with 20 ml buffer D, before being eluted 3 times with 3 ml of 20 mM glutathione or 150 mM imidazole in buffer D. For BARD1 124-270 , the His 6 -SUMO tag was cleaved by the Ulp1 protease by an overnight incubation at 4°C.

Techniques: Mutagenesis

Journal: Nature

Article Title: Promotion of RAD51-mediated homologous DNA pairing by BRCA1-BARD1

doi: 10.1038/nature24060

Figure Lengend Snippet: a, Immunoprecipitation to test BARD1WT and BARD1AAE for RAD51 association upon MMC treatment. The asterisk denotes a non-specific band. b, Schematic of the DR-GFP reporter assay (upper). Results obtained with cells expressing BARD1WTres or BARD1AAEres upon treatment with BARD1 siRNA or control siRNA (siCtrl) (bottom). Data are means ± s.d., n=3. c, Schematic of the CRISPR/Cas9 gene targeting assay (upper). Results obtained with cells expressing BARD1WTres or BARD1AAEres upon treatment with BARD1 siRNA or siCtrl. Data are means ± s.d., n=3. d, Clonogenic survival of cells expressing BARD1WTres or BARD1AAEres upon Olaparib or MMC treatment. Data are means ± s.d., n=3. EV, empty vector. P values were calculated using two-way ANOVA and multiple comparisons were corrected by the Bonferroni method. **, P<0.01.

Article Snippet: After centrifugation (100,000 × g for 90 min), the clarified lysate was incubated with 2 ml Glutathione Sepharose 4 Fast Flow resin (GE Healthcare; for GST-BARD1 123-162 ) or Ni-NTA resin (GE healthcare; for BARD1 124-270 ) for 2 h. The affinity resin was transferred to a glass column (1.5 × 15 cm), washed with 20 ml buffer D, before being eluted 3 times with 3 ml of 20 mM glutathione or 150 mM imidazole in buffer D. For BARD1 124-270 , the His 6 -SUMO tag was cleaved by the Ulp1 protease by an overnight incubation at 4°C.

Techniques: Immunoprecipitation, Reporter Assay, Expressing, CRISPR, Plasmid Preparation

Journal: Nature

Article Title: Promotion of RAD51-mediated homologous DNA pairing by BRCA1-BARD1

doi: 10.1038/nature24060

Figure Lengend Snippet: Aside from a co-operative role with PALB2-BRCA2 in RAD51 presynaptic filament assembly (green arrows), our work has revealed a function of BRCA1-BARD1 in the promotion of homologous DNA pairing (red arrows). Previous studies have provided evidence that BRCA1-BARD1 antagonizes 53BP1 in DNA end resection (green blocks) and promotes MRN/CtIP activity (green arrows), and for a role of the complex in cell cycle checkpoint regulation (green arrows).

Article Snippet: After centrifugation (100,000 × g for 90 min), the clarified lysate was incubated with 2 ml Glutathione Sepharose 4 Fast Flow resin (GE Healthcare; for GST-BARD1 123-162 ) or Ni-NTA resin (GE healthcare; for BARD1 124-270 ) for 2 h. The affinity resin was transferred to a glass column (1.5 × 15 cm), washed with 20 ml buffer D, before being eluted 3 times with 3 ml of 20 mM glutathione or 150 mM imidazole in buffer D. For BARD1 124-270 , the His 6 -SUMO tag was cleaved by the Ulp1 protease by an overnight incubation at 4°C.

Techniques: Activity Assay