Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Surface view of the 3D reconstruction. Two halves were colored yellow and cyan, representing two potential monomers although the exact boundary is unknown. ( b-g ) Antibody labeling against C-terminal Flag tag ( b-d ) and BRC repeats ( e-g ). In ( b ) and ( e ), raw particles with antibody are circled. ( c ) and ( f ) Reprojections along the same orientations of ( b) and ( e) . ( d ) and ( g ) 3D reconstructions viewed along the same directions as ( b) and ( e) , with antibody locations represented by spheres. ( h ) and ( i ) Top and side views of BRCA2 with antibody locations colored (Flag tag- blue, BRC – magenta). Magnification bars in all single particle images represent 100 Å.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Antibody Labeling, FLAG-tag, Single Particle

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) and ( b ) Side and top surface views of the 3D reconstruction. ( c ) Antibody labeling against RAD51. Left: individual particles with antibody circled. Middle: corresponding reprojections from the BRCA2-RAD51 reconstruction. Right: surface view along the same direction with antibody locations indicated with spheres. ( d ) Cylinders representing antibody locations defined from individual particles (upper). These intersect at the density regions on the outer rim connecting the two halves (lower, orange surface).

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Antibody Labeling

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Overlay of BRCA2 dimer (yellow and cyan) and BRCA2-RAD51 (pink mesh) highlighting the differences in their shape. ( b ) Rearranged BRCA2 dimer fitted into the BRCA2-RAD51 complex. ( c ) as in (b). Four RAD51 monomers (orange ribbon) were fitted into the additional density in BRCA2-RAD51 not accounted for by BRCA2 density. ( d ) Four RAD51 monomers, arranged as in filaments. (e) Histogram of mass measurement of BRCA2-RAD51 complex using STEM, showing peaks at 800 kD and 1200 kD, corresponding to BRCA2 dimer and BRA2 dimer binding to 8-10 RAD51.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Mass Measurement, Binding Assay

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Gel-shift assay showing the binding of BRCA2 to 5′- 32 P-labeled ssDNA substrates ranging from 20 to 100 nt. DNA was detected by autoradiography. ( b ) Images of individual particles of BRCA2 bound to gapped DNA (duplex arms are indicated in orange). Magnification bars represent 100 Å. ( c-e ) Electron microscopic visualization of RAD51-ssDNA filaments, BRCA2-ssDNA complexes, and BRCA2-RAD51-ssDNA complexes, as indicated. ( f ) Localization of BRCA2 in BRCA2-RAD51-ssDNA complexes by immunogold labeling. ( g ) Visualization of BRCA2-RAD51 filaments formed with 5′-gold particle labeled ssDNA. Magnification bars represent 100 nm.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Gel Shift, Binding Assay, Labeling, Autoradiography

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) and ( b ) Effect of BRCA2 on the number of RAD51-ssDNA nucleation events, as determined by electron microscopy. Inserts show enlargements of RAD51 filaments. ( c-d ) Quantification of RAD51-ssDNA filament length ( c ) and nucleation events ( d ) in the presence (blue) or absence (orange) of BRCA2, as determined by measurement of images shown in (n = 314) and 4 e (n = 332), n = number of RAD51 filaments. In total, 204 (RAD51-ssDNA) and 149 (BRCA2-RAD51-ssDNA) randomly collected grid areas were quantified. P-values (P < 0.0001) were determined using a two-tailed t test, error bars represent SD. ( e ) BRCA2-RAD51-ssDNA complexes visualized as multiple distinct filament nucleation sites on the same ssDNA molecule (arrowed). Magnification bars represent 100 nm.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Electron Microscopy, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Structure and Mechanism of Action of the BRCA2 Breast Cancer Tumor Suppressor

doi: 10.1038/nsmb.2899

Figure Lengend Snippet: ( a ) Crystal structure of RPA bound to 30 nt ssDNA, showing a compact configuration and bending of the ssDNA into a U-shape. DNA binding domains of BRCA2 could adapt similar conformations. The polarity of the ssDNA is indicated. The two RPA molecules, related by 2-fold symmetry, could represent the DNA binding domains in the BRCA2 dimer as indicated below. ( b ) The DNA binding domains (1,2,3,4) of BRCA2 are depicted in similar conformations as those shown for RPA in (a), such that ssDNA could simultaneously bind to domains 3-4 (OB2-OB3) at the 5′ end (left hand side) of one BRCA2 monomer while domains 1-2 (alpha-helical domain and OB1) at the 3′ end (right hand side) of the second monomer. Two sets of RAD51 molecules bind the BRCA2 dimer in opposing directions. Only one set can be productive in ssDNA binding. ( c ) Model for filament formation and elongation using multiple BRCA2-RAD51 nucleation sites with BRCA2 acting as a molecular chaperone for RAD51.

Article Snippet: For BRCA2 FLAP we used anti-Flag M2-HRP antibody (Sigma-Aldrich) and the anti-BRCA2 OP95 mouse antibody (Merck Millipore, Ab-1).

Techniques: Binding Assay

Journal: The Journal of Cell Biology

Article Title: ATP-dependent and independent functions of Rad54 in genome maintenance

doi: 10.1083/jcb.201011025

Figure Lengend Snippet: The Rad54 protein, but not its ATPase activity, affects Rad51 recruitment to sites of DSBs. Accumulation of DSB repair proteins at α particle–induced DSB tracks. (A) Localization of Rad54 to the α particle–induced double-stranded break colocalizing with DSB marker γH2Ax. Bar, 5 µm. (B) RAD54 protein levels in U2Os cells transfected with indicated siRNAs. Cell lysates were analyzed by immunoblotting with antibodies against RAD54. Equal sample loading was verified by the equal presence of nonspecific bands. (C) Quantification of accumulation of Nbs1, RPA, Rad51, and BRCA2 at α particle–induced tracks of DSBs 0, 5, 15, and 60 min after irradiation in the presence or absence Rad54. U2Os cells were stained for either γH2Ax (Nbs1 and Rad51) or 53BP1 (RPA and Brca2) as a DSB marker and for one of the indicated repair proteins at t = 0, 5, 15, and 60 min after irradiation. t = 0 indicates the first time point after α particle irradiation. Graphs represent mean percentage of positive DSB tracks with a repair protein. 100 cells containing α particle–induced tracks were scored per experiment. Error bars represent the range of percentages obtained from three independent experiments. (D) Quantification of Rad51 accumulation at DSB sites 0, 5, 15, and 60 min after α particle irradiation in Rad54 +/+ , Rad54 −/− , Rad54 wt-GFP/− , and Rad54 K189R-GFP/− ES cells. Graphs represent mean percentage of Rad51-positive tracks per γH2Ax track. 100 cells containing damage induced by α particles were scored per experiment. Error bars represent the range of percentages obtained from two independent experiments.

Article Snippet: The primary antibodies used in this study were: anti-Rad51 (rabbit polyclonal; ), anti-Rad54 (rabbit polyclonal; ), anti-γH2AX (Millipore), anti-53BP1 (rabbit polyclonal; Novus Biologicals), anti-Rad54 (goat polyclonal, D-18; Santa Cruz Biotechnology, Inc.), anti-NBS1 (goat polyclonal, C-19; Santa Cruz Biotechnology, Inc.), anti-RPA34 (mouse monoclonal, Ab-2; Oncogene), and anti-BRCA2 (mouse monoclonal, Ab-1; EMD).

Techniques: Activity Assay, Marker, Transfection, Western Blot, Irradiation, Staining

Journal: Cancer Research

Article Title: BRCA2 and Nucleophosmin Coregulate Centrosome Amplification and Form a Complex with the Rho Effector Kinase ROCK2

doi: 10.1158/0008-5472.can-10-0030

Figure Lengend Snippet: Figure 1. Identification of NPM and ROCK2 as candidates for BRCA2-associated centrosomal proteins and binding of NPM to BRCA2 in vivo. A, identification of NPM and ROCK2 as candidates for BRCA2-associated centrosomal proteins by immunoprecipitation and mass spectrometric screening. Centrosomes from HeLa S3 cells were isolated (lane 1), and the centrosomal components were immunoprecipitated with anti-BRCA2 antibody. The immunoprecipitates were subjected to SDS-PAGE and visualized by silver staining (lane 3). The protein bands specific to BRCA2 association were analyzed with LC/MS/MS as summarized. Normal IgG did not precipitated NPM or ROCK2 (lane 2). B, in vivo physical interaction between exogenously expressed NPM and BRCA2. COS-7 cells were transiently cotransfected with the combinations of either a FLAG-BRCA2 expression vector or the empty one, and either an HA-NPM expression vector or the empty one. The cell lysates were immunoprecipitated with either anti-HA or anti-FLAG antibody, and the immunoprecipitates were analyzed by immunoblotting with either anti-FLAG (the top 3 panels) or anti-HA antibody (the bottom 3 panels). C and D, binding of endogenous NPM and BRCA2. C, the immunoprecipitates were detected with anti-BRCA2 antibody. Top, lysates from COS-7 cells (lane1) were immunoprecipitated with either anti-BRCA2 antibody (lane 2) or normal mouse IgG (lane 3). Bottom, lysates (lane 1) were immunoprecipitated with either normal mouse IgM (lane 2) or anti-NPM antibody (lane 3). D, The immunoprecipitates were detected with anti-NPM antibody. Top, lysates from COS-7 cells (lane 1) were immunoprecipitated with either anti-NPM antibody (lane 2) or normal mouse IgM (lane 3). Bottom, lysates (lane 1) were immunoprecipitated with either anti-BRCA2 antibody (lane 2) or normal mouse IgG (lane 3). IB, immunoblotting; IP, immunoprecipitation.

Article Snippet: The antibodies used in this study were anti-ROCK2 polyclonal antibody (Upstate Biotechnology; 07-443), anti-NPM/ B23 mouse monoclonal antibody (Chemicon; MAB4500), antiNPM/B23 mouse monoclonal antibody (Santa Cruz; FC-8791), anti-B23 monoclonal antibody (Sigma; B0556), anti–phosphoNPM (anti–p-NPM; Thr199) rabbit polyclonal antibody (Cell Signaling Technology; 3541), anti-mouse IgM antibody (Sigma; M8644), anti-BRCA2 mouse monoclonal antibody (Oncogene; Ab-1), anti-BRCA2 rabbit polyclonal antibody (Santa Cruz; H299), anti-FLAG monoclonal antibody (Sigma; F3165), antig-tubulin rabbit antibody (Sigma; T3559), anti-g-tubulin monoclonal antibody (Santa Cruz; C-11), anti-centrin2 goat polyclonal antibody (Santa Cruz; N-17), anti-centrin1/2 rabbit polyclonal antibody (Santa Cruz; H-40), and anti-HA rat monoclonal antibody (Roche; clone 3F10).

Techniques: Binding Assay, In Vivo, Immunoprecipitation, Isolation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Expressing, Plasmid Preparation, Western Blot

Journal: Cancer Research

Article Title: BRCA2 and Nucleophosmin Coregulate Centrosome Amplification and Form a Complex with the Rho Effector Kinase ROCK2

doi: 10.1158/0008-5472.can-10-0030

Figure Lengend Snippet: Figure 2. Binding of ROCK2 to BRCA2. A and B, binding of exogenous FLAG-BRCA2 and endogenous ROCK2. COS-7 cells were transiently transfected with either full-length FLAG-BRCA2 expression vector (lane 1) or the empty one (lane 2), respectively. A, The cell lysates (top), the immunoprecipitates with anti-FLAG (middle) and anti-ROCK2 antibodies (bottom) were analyzed by immunoblotting with anti-FLAG antibody. B, the cell lysates (top), the immunoprecipitates with anti-ROCK2 (middle) and anti-FLAG antibodies (bottom) were analyzed by immunoblotting with anti-ROCK2 antibody. C and D, binding of endogenous BRCA2 and ROCK2. C, the immunoprecipitates were detected with anti-BRCA2 antibody. Top, lysates from COS-7 cells (lane 1) were immunoprecipitated with either anti-BRCA2 antibody (lane 2) or the normal mouse IgG (lane 3). Bottom, lysates (lane 1) were immunoprecipitated with either anti-ROCK2 antibody (lane 2) or normal mouse IgG (lane 3). D, the immunoprecipitates were detected with anti-ROCK2 antibody. Top, lysates from COS-7 cells (lane 1) were immunoprecipitated with either anti-ROCK2 antibody (lane 2) or the normal mouse IgG (lane 3). Bottom, lysates (lane 1) were immunoprecipitated with either anti-BRCA2 antibody (lane 2) or normal mouse IgG (lane 3). IB, immunoblotting; IP, immunoprecipitation.

Article Snippet: The antibodies used in this study were anti-ROCK2 polyclonal antibody (Upstate Biotechnology; 07-443), anti-NPM/ B23 mouse monoclonal antibody (Chemicon; MAB4500), antiNPM/B23 mouse monoclonal antibody (Santa Cruz; FC-8791), anti-B23 monoclonal antibody (Sigma; B0556), anti–phosphoNPM (anti–p-NPM; Thr199) rabbit polyclonal antibody (Cell Signaling Technology; 3541), anti-mouse IgM antibody (Sigma; M8644), anti-BRCA2 mouse monoclonal antibody (Oncogene; Ab-1), anti-BRCA2 rabbit polyclonal antibody (Santa Cruz; H299), anti-FLAG monoclonal antibody (Sigma; F3165), antig-tubulin rabbit antibody (Sigma; T3559), anti-g-tubulin monoclonal antibody (Santa Cruz; C-11), anti-centrin2 goat polyclonal antibody (Santa Cruz; N-17), anti-centrin1/2 rabbit polyclonal antibody (Santa Cruz; H-40), and anti-HA rat monoclonal antibody (Roche; clone 3F10).

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation