Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic BRCA2 mutations in the DBD affect DSS1 binding and nuclear localization. ( A ) List of pathogenic (red) or benign (blue) mutations in the DBD analysed in this study with a schematic of the expression construct, GFP-BRCA2 CT . ( B and C ) Interaction between the DBD mutants and DSS1. Wild-type or mutant forms of GFP-BRCA2 CT were transiently expressed with Myc-DSS1 in 293T cells. Interaction was detected by anti GFP-IP and western blotting with anti-Myc antibody. The graph ( C ) shows DSS1-binding activity of the mutants normalised to the WT value. Binding activity was assessed by quantifying the intensity of the Myc-DSS1 bands pulled-down by IP. Data are presented as mean ± s.e. from three repeats. Statistical significance was assessed by 1-way ANOVA test, followed by Dunnett's multiple comparison test (compared to WT). (D and E) Nucleocytoplasmic distribution of the DBD mutants. HeLa cells transiently expressing GFP-BRCA2 CT fragments were fixed with PFA before imaging. The graph ( E ) shows the GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.). More than 100 cells per sample were analysed. Statistical significance was assessed by the Kruskal-Wallis test, followed by Dunnett's multiple comparison test (compared to WT). A representative result from two independent experiments is shown. ( D ) shows representative microscopic images. Scale bar, 10μm.

Article Snippet: HeLa Kyoto (from the laboratory of Jonathan Pines) and 293T (ATCC) cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and maintained at 37°C with 5% CO 2 .

Techniques: Binding Assay, Expressing, Construct, Mutagenesis, Western Blot, Activity Assay, Comparison, Imaging

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: The BRCA2-DSS1 interaction antagonizes intracellular BRCA2 oligomerization. ( A ) Self-oligomerization of BRCA2 CT is suppressed by Myc-DSS1. Wildtype or D2723H mutant forms of BRCA2 CT , tagged with either GFP or mCherry, were transiently expressed in 293T cells with or without Myc-DSS1. Interaction was detected by anti GFP-IP and western blotting for mCherry-BRCA2 CT and endogenous BRCA2. ( B ) Self-oligomerization of BRCA2 CT fragments in the BRCA2 Knock-out (KO) HeLa cells. BRCA2 CT fragments tagged with either GFP or mCherry were co-expressed in BRCA2 KO HeLa cells. Their interaction was detected by anti GFP-IP and western blotting for mCherry-BRCA2 CT . GFP vector alone serves as a negative control. ( C ) GST-pull down assay. Purified GST-BRCA2 CT fragments bound to the Glutathione Sepharose 4B beads were incubated with purified GFP-BRCA2 CT fragments. The beads were washed, and bound proteins were eluted, before separation by SDS-PAGE. Interactions were detected by western blotting for GFP-BRCA2 CT . GST alone serves as a negative control. ( D ) Native gel electrophoresis of GFP-BRCA2 CT . Extracts from HeLa cells transiently expressing GFP-BRCA2 CT forms with or without Myc-DSS1 were analysed by native gel electrophoresis. The bands corresponding to GFP-BRCA2 CT oligomers, GFP-BRCA2 CT /Myc-DSS1 complex and GFP-BRCA2 CT are marked with arrows.

Article Snippet: HeLa Kyoto (from the laboratory of Jonathan Pines) and 293T (ATCC) cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and maintained at 37°C with 5% CO 2 .

Techniques: Mutagenesis, Western Blot, Knock-Out, Plasmid Preparation, Negative Control, Pull Down Assay, Purification, Incubation, SDS Page, Nucleic Acid Electrophoresis, Expressing