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: Molecular Cancer

Article Title: CHK1 inhibition as a strategy for targeting fanconi anemia (FA) DNA repair pathway deficient tumors

doi: 10.1186/1476-4598-8-24

Figure Lengend Snippet: Analysis of the mechanism of cell death and the cell cycle effect of CHK1 inhibition in FA pathway deficient cells . (A) An annexin V/PI staining cell death assay to assess mode of cell death in HeLa cells treated with siRNA targeting GFP , FANCA , and Gö6976. Twenty-four hrs after siRNA transfection, the cells were treated with 500 nM Gö6976 or DMSO for an additional 48 hrs. Cells were then collected and subjected to annexin V/PI staining. Cells were also treated with cisplatin 10 μM for 24 hrs as a positive apoptotic control. (B) FA pathway knockdown activates CHK1 and accumulation of cells in the G2 phase of the cell cycle. Western blots measuring phosphorylation of CDC25C, CHK1, and monoubiquitination of FANCD2 in HeLa cells treated with control siRNA (lanes 1 and 2), siRNA targeting FANCA (lanes 3 and 4), and siRNA targeting BRCA2 (lanes 5 and 6). Cells were either treated for 24 hrs with Gö6976 (lanes 2, 4, 6) or DMSO control (lanes 1, 3, 5). Membranes were probed for vinculin to ensure equal loading. (C) Cell cycle analysis of HeLa cells treated with control GFP targeted siRNA, FANCA targeted siRNA or BRCA2 targeted siRNA followed by Gö6976 or DMSO control for 24 hrs. The mitotic population was assessed by measuring histone H3 phosphorylation by flow cytometry. Populations of cells at G1 (2N), S (between 2N and 4N), G2/M (4N), G2 (4N with no histone H3 staining), and M (4N with phosphohistone H3 staining) are given as a percentage of total cell population. Values are calculated from duplicate experiments.

Article Snippet: Membranes were then probed with the following antibodies: anti-FANCD2 (1:1000; Santa Cruz Cat#sc-20022), anti-FANCA (1:1000; [ ]) anti-FANCF (1:1000, [ ], anti-BRCA2 (1:1000, Calbiochem Cat#OP95), anti-phosphoH2AX Ser 139 (1:2000; Upstate Cell Signaling Solutions Cat#07-164), anti-phopsho-317-CHK1 (1:500; Cell Signaling Cat#2344), anti-CHK1(G-4) (Santa Cruz Cat#8480), antiphospho-CDC25C (1:1000; Cell Signaling Cat#4901), anti-GAPDH (1:3000; Abcam Cat#ab9484), and anti-vinculin (1:2000; Santa Cruz Cat#sc25336).

Techniques: Inhibition, Staining, Transfection, Western Blot, Cell Cycle Assay, Flow Cytometry

Journal: EMBO Reports

Article Title: CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage

doi: 10.15252/embr.201948920

Figure Lengend Snippet: U2OS cells were transfected with control (siLuc) or BRCA2‐targeting siRNA (siBRCA2). After 48 h, cells were either mock‐treated or treated with 10 μM XL413 for 30 min and labelled consecutively with CldU (red) and IdU (green), followed by treatment with 4 mM HU for a further 5 h. Representative Western blot of indicated proteins obtained from whole‐cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413. The ratios of IdU/CldU tract length are plotted. The box extends from the 25 th to the 75 th percentile with the line in the box representing the median. Whiskers indicate the 10–90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analysed for each condition. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: Student's t ‐test; ns, not significant; ** P < 0.01; *** P < 0.001 Electron micrographs of representative replication forks from U2OS cells: A reversed fork is labelled: P = parental strand; D = daughter strand; and R = regressed arm; the four‐way junction at the reversed fork is magnified in the inset. The red arrows indicate single‐stranded DNA at the fork. Scale bar is 200 nm (= 460 bp) and 100 nm (= 230 bp) in the inset. Frequency of reversed replication forks isolated from mock‐depleted (siLuc) and BRCA2‐depleted (siBRCA2) U2OS cells upon 5‐h treatment with 4 mM HU in the presence or absence of 10 μM XL413. The number of replication intermediates analysed is indicated in parentheses. Similar results were obtained in two independent experiments ( Appendix Table S1 ). Amount of ssDNA length at the junction (red arrows in Fig D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 h in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork, and the value is displayed above the plot. Source data are available online for this figure.

Article Snippet: Primary antibodies diluted in blocking buffer (TBST, 5% milk) included RPA2 (1:1,000, NA19L; Calbiochem), Histone H2A (1:1,000, 07‐146; Upstate), MRE11 (1:1,000, NB100‐142; Novus), EXO1 (1:1,000, A302‐640A; Bethyl Laboratories), BRCA2 (1:2,000, A303‐434A; Bethyl Laboratories; 1:500, OP 95; EMD Millipore Ab‐1), Histone H3 (1:1,000, A300‐823A; Bethyl Laboratories), CDC7 (1:1,000, K0070‐3; MBL), CHK1 (1:1,000, SC‐8408; Santa Cruz Biotech) and CDC45 (1:5, a gift from Broderick et al ).

Techniques: Transfection, Control, Western Blot, Inhibition, Standard Deviation, Isolation

Journal: EMBO Reports

Article Title: CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage

doi: 10.15252/embr.201948920

Figure Lengend Snippet: U2OS or AS‐CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2)‐targeting siRNAs and then either mock‐treated or treated with 4 mM HU for 5 h in the presence or absence of 10 μM XL413 or 3MB‐PP1, respectively. HU was then washed off and nocodazole was added to the medium in order to complete one round of DNA synthesis. Metaphase spreads were prepared and analysed. A Representative metaphase spread from BRCA2‐depleted U2OS cells treated with HU. Scale bar = 5 μm. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*). B, C The graphs show the average number of chromatid breaks per spread. In each experiment, 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance was assessed by Student's t ‐test (** P ˂ 0.01, *** P < 0.001). D, E U2OS and AS‐CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2)‐targeting siRNAs. Forty‐eight hours post‐transfection, cells were either mock‐treated or treated with 10 μM XL413 or 10 μM 3MB‐PP1, respectively, for 24 h with nocodazole added for the last 16 h of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment, 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance was assessed by Student's t ‐test (* P < 0.05, *** P < 0.001). Source data are available online for this figure.

Article Snippet: Primary antibodies diluted in blocking buffer (TBST, 5% milk) included RPA2 (1:1,000, NA19L; Calbiochem), Histone H2A (1:1,000, 07‐146; Upstate), MRE11 (1:1,000, NB100‐142; Novus), EXO1 (1:1,000, A302‐640A; Bethyl Laboratories), BRCA2 (1:2,000, A303‐434A; Bethyl Laboratories; 1:500, OP 95; EMD Millipore Ab‐1), Histone H3 (1:1,000, A300‐823A; Bethyl Laboratories), CDC7 (1:1,000, K0070‐3; MBL), CHK1 (1:1,000, SC‐8408; Santa Cruz Biotech) and CDC45 (1:5, a gift from Broderick et al ).

Techniques: Transfection, Control, DNA Synthesis