Journal: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: Dual C-terminal and N-terminal BRCA2 immunohistochemistry (IHC) of hereditary breast cancers (A and B, respectively) and of sporadic breast cancers (C and D, respectively). Magnification is (left) ×20 and (right) ×100 for all panels. The upper panel for each pair is C-terminal IHC, and the lower panel is N-terminal IHC. (A) Samples are from a BRCA2-mutant cancer with 7231del5. Top panel stained with C-terminal BRCA2 antibody, and second panel stained with N-terminal BRCA2 antibody. Only a lymphocyte in the top panel stains. (B) Samples are from a patient with a 9654delTT BRCA2 mutation at similar magnifications and similar staining. (C and D) The paired panels are from adjacent sections of the same sporadic breast cancer sample. Note the similar nuclear staining with both antibodies for the sporadic cancer samples.

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: Immunohistochemistry, Mutagenesis, Staining

Journal: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: Immunohistochemistry with 575A15 C-terminal monoclonal antibody on normal breast epithelial lobules. Upper panels: untreated antibody. Middle panels: antibody mixed with excess of immunizing peptide (BRCA2 amino acids 3284 to 3294: TFVSPAAKAGG). Lower panels: antibody mixed with excess of control BRCA2 peptide (BRCA2 amino acids between 3300 and 3400, obtained from Abcam (Cambridge MA). Magnification is (left) ×20 and (right) ×100.

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: Immunohistochemistry

Journal: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: MCF7 cells were cultured for 48 hours in 10% charcoal-stripped serum/phenol red–free DMEM and were treated with 10 nmol/L estrogen for 5 and 30 minutes and for 1, 2, 4, and 24 hours. Samples were blotted with the 575A15 BRCA2 C-terminal monoclonal antibody. The 440 kDa band is the only band on the blot.

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: Cell Culture

Journal: Journal of Clinical Oncology

Article Title: Detecting BRCA2 Protein Truncation in Tissue Biopsies to Identify Breast Cancers That Arise in BRCA2 Gene Mutation Carriers

doi: 10.1200/JCO.2008.20.5211

Figure Lengend Snippet: BRCA2 Mutations Identified in Patients With Breast Cancer

Article Snippet: The N-terminal antibody used was from R&D systems MAB2476 (a commercially available mouse monoclonal antibody directed against an E coli –derived recombinant human BRCA2 protein that spans amino acids 1-200), which was used at 3 μg/mL.

Techniques: Mutagenesis

Journal: Molecular Genetics & Genomic Medicine

Article Title: BRCA2 c.8827C>T pathogenic mutation in a consanguineous Chinese family with hereditary breast cancer

doi: 10.1002/mgg3.1411

Figure Lengend Snippet: Pedigree diagram of a consanguineous Chinese family with BRCA2 mutations. The pedigree shows two generations (I and II). Filled symbols indicate the patients, while blank symbols indicate unaffected members. Diagonal lines through each square or circle signify the deceased family members. The black arrow denotes the proband (II: 7). PC, prostate cancer; BC, breast cancer; BBC, bilateral breast cancer; +, proven BRCA2 c.8827C>T mutation; ‐, proven normal at BRCA2 c.8827C; b., year of birth; diag. age, cancer detection age; d. age, death age

Article Snippet: Membranes were then incubated with antibody against BRCA2 (1:500; Thermo Fisher Scientific, Inc.) and β‐actin (1:500; Thermo Fisher Scientific, Inc.) overnight at 4°C before washing three times in TBST.

Techniques: Mutagenesis

Journal: Molecular Genetics & Genomic Medicine

Article Title: BRCA2 c.8827C>T pathogenic mutation in a consanguineous Chinese family with hereditary breast cancer

doi: 10.1002/mgg3.1411

Figure Lengend Snippet: (a) The nonsense BRCA2 c.8827C>T mutation identified in proband (II: 7) in this family through next‐generation sequencing platform. (b) BRCA2 c.8827C>T mutation locating in the DNA‐binding domain of its coding protein. BRC, breast cancer; DBD, DNA‐binding domain; NLS, nuclear localization signal; TAD, transactivation domain

Article Snippet: Membranes were then incubated with antibody against BRCA2 (1:500; Thermo Fisher Scientific, Inc.) and β‐actin (1:500; Thermo Fisher Scientific, Inc.) overnight at 4°C before washing three times in TBST.

Techniques: Mutagenesis, Next-Generation Sequencing, Binding Assay

Journal: Molecular Genetics & Genomic Medicine

Article Title: BRCA2 c.8827C>T pathogenic mutation in a consanguineous Chinese family with hereditary breast cancer

doi: 10.1002/mgg3.1411

Figure Lengend Snippet: Real‐time PCR result (a) and Western blot image (b) showing the expressions of BRCA2 mRNA and BRCA2 protein in individuals with or without BRCA2 c.8827C>T mutation, respectively. WT, wild‐type

Article Snippet: Membranes were then incubated with antibody against BRCA2 (1:500; Thermo Fisher Scientific, Inc.) and β‐actin (1:500; Thermo Fisher Scientific, Inc.) overnight at 4°C before washing three times in TBST.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Mutagenesis

Journal: Nucleic Acids Research

Article Title: p21 promotes error-free replication-coupled DNA double-strand break repair

doi: 10.1093/nar/gks612

Figure Lengend Snippet: An increased CDK-mediated BRCA2 S3291 phosphorylation in p21 −/− cells. ( A and B ) HCT116 p21 +/+ and p21 −/− cells were incubated in the absence or presence of 20 or 40 nM MMC for 16 h. Whole-cell lysates were prepared, and resolved proteins were immunoblotted with anti-BRCA2, anti-BRCA2 pS3291 and anti-lamin A antibodies. (B) Band intensities from (A) were quantified using ImageJ and the ratios of BRCA2 pS3291 to BRCA2 plotted. ( C and D ) p21 −/− cells were incubated in the absence or presence of 10 and 20 nM MMC and 3 µM roscovitine (Rosc.) for 16 h. Metaphase spreads were prepared, and chromosome aberrations, including gaps and breaks (C) and complex chromosome aberrations (D), including radial formations, were scored. At least 80 metaphases were scored per treatment, and this experiment was repeated twice with similar findings. Error bars represent standard errors of the means. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Membranes were immunoblotted with mouse monoclonal antisera against BRCA2 (OP95; Calbiochem/EMD Chemicals), DNA-PK CS (Ab-2/25-4; Thermo Scientific), γH2AX (JBW301; Millipore) and α-tubulin (Ab-2; LabVision), and rabbit polyclonal antisera BRCA2 pS3291 , DNA-PK CS pS2056 (ab18192; Abcam), p21 (N-20; Santa Cruz Biotechnology) and MRE11 (NB100-142; Novus Biologicals).

Techniques: Phospho-proteomics, Incubation

Journal: Nucleic Acids Research

Article Title: p21 promotes error-free replication-coupled DNA double-strand break repair

doi: 10.1093/nar/gks612

Figure Lengend Snippet: Proposed model for the role of p21 in replication-coupled DNA double-strand break repair. Following exposure to DNA-damaging agents that block DNA replication fork progression, p21 physically binds to, and inhibits, CDK, thereby preventing phosphorylation of BRCA2 on S3291. RAD51 nucleoprotein filaments assemble on ssDNA and are stabilized through interaction with the unphosphorylated carboxy-terminus of BRCA2, thereby promoting HR repair. In the absence of p21, unrestricted CDK activity leads to the constitutive phosphorylation of BRCA2 on S3291. S3291 phosphorylation precludes binding of RAD51 to the carboxy-terminus of BRCA2, resulting in destabilization of RAD51 nucleoprotein filaments. Deprotected ssDNA now becomes susceptible to opportunistic and inadvertent MRE11 nuclease activity, leading to increased error-prone NHEJ and chromosome instability.

Article Snippet: Membranes were immunoblotted with mouse monoclonal antisera against BRCA2 (OP95; Calbiochem/EMD Chemicals), DNA-PK CS (Ab-2/25-4; Thermo Scientific), γH2AX (JBW301; Millipore) and α-tubulin (Ab-2; LabVision), and rabbit polyclonal antisera BRCA2 pS3291 , DNA-PK CS pS2056 (ab18192; Abcam), p21 (N-20; Santa Cruz Biotechnology) and MRE11 (NB100-142; Novus Biologicals).

Techniques: Blocking Assay, Phospho-proteomics, Activity Assay, Binding Assay

Journal: Nature communications

Article Title: Elongator and codon bias regulate protein levels in mammalian peripheral neurons.

doi: 10.1038/s41467-018-03221-z

Figure Lengend Snippet: Fig. 4 Depleted levels of BRCA2 are correlated with elevated levels of DNA damage. a RT-qPCR confirms normal levels of Brca2 transcript (P = 0.06). b–d Quantitative immunohistochemistry confirms decreased levels of BRCA2 protein (P < 0.001). e–g Longer comet tails in the CKO show fragmented DNA (P = 0.009); error bars denote SD in a and SEM in d, g; scale bar: b, c 40 μm; e, f 12 μm

Article Snippet: Primary antibodies included the following: BRCA2 (Biorbyt, orb10203, 1:25), VCAN (Abcam, ab177480, 1:100), S-100 (Agilent Technologies, Z0311, 1:400), Tuj1 (Biolegend, 801202, 1:1,000), and Histone H2A (Biorbyt, orb127582, 1:200).

Techniques: Quantitative RT-PCR, Immunohistochemistry

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: Clustering of pathogenic BRCA2 missense mutations within the DBD. ( A ) Clinical significance of BRCA2 mutations by mutation type. The graph shows 9085 variants classified by molecular consequence (mutation type) and clinical significance, out of a total of 12 684 BRCA2 variants listed in the ClinVar database (version of 2020.07.27). ( B ) Clinical significance of 143 BRCA2 missense mutations reviewed by the expert panel are shown. Pathogenic mutations clustered in the DBD are marked with a blue circle. The schematic shows human BRCA2 protein structure. Amino acid residue numbers are marked. HD, helical domain: OB, oligonucleotide/oligosaccharide binding; TD, tower domain; NLS, nuclear localisation signal. ( C ) Location of each pathogenic mutation within the BRCA2 DBD.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Mutagenesis, Residue, Binding Assay

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: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

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: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

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

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 DBD mutants introduced into the endogenous BRCA2 gene cause cytosolic mis-localization and disable DNA repair by HDR. ( A ) Subcellular fractionation of BRCA2 mutant cell lines, HeLa-W2626C and HeLa-D2723H. Two independent clones for each mutation were used. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. MEK2 is a cytoplasmic marker. ( B ) mClover Lamin A HDR assay. Cells transfected with Lamin A-targeting sgRNA and mClover Lamin A donor constructs as shown in the schematic were analysed for mClover Lamin A-positive cells. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was tested by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Fractionation, Mutagenesis, Clone Assay, Western Blot, Marker, Transfection, Construct, Comparison

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 DBD mutants exhibit defective nuclear import. ( A ) Nuclear import assay using BRCA2 CT tagged with photoactivable GFP (paGFP). HeLa cells expressing wildtype or mutant forms of paGFP-BRCA2 CT were monitored by live imaging. As shown in the schematic, photoactivation of a cytosolic area (red square) was followed by measurement of fluorescence intensity in the designated area in the cytosol (red square) and the nucleus (blue square). Plots show the relative fluorescence intensity (mean ± s.e.), normalized to the maximum intensity in each cell. 8–10 cells per sample were analysed. Black arrows mark the timing of photoactivation. ( B ) HeLa cells expressing paGFP-BRCA2 CT W2626C with or without Myc-DSS1 were monitored by live imaging after cytosolic photoactivation and analysed as shown in (A). ( C ) Efficiency of DSS1 depletion by siRNA transfection. Relative level of DSS1 mRNA measured by RT-qPCR assay, a result from technical triplicate is shown. Statistical significance was assessed by paired t -test. ( D and E ) Nucleocytoplasmic distribution of the GFP-BRCA2 CT fragment after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 24 h before transfecting with the GFP-BRCA2 CT construct. Nucleocytoplasmic distribution was analysed as described in Figure . 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 unpaired t-test. A representative result from three independent experiments is shown. Representative microscopic images are shown in (D). Scale bar, 10 μm. ( F ) Subcellular fractionation of HeLa cells after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 48 h before fractionation. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. The nuclear/cytoplasmic ratio of BRCA2 expression is shown. MEK2 is a cytoplasmic marker. Total cell extracts are also shown. A representative result from two independent experiments is shown.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Expressing, Mutagenesis, Imaging, Fluorescence, Transfection, Quantitative RT-PCR, Control, Construct, Fractionation, Western Blot, Marker

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: DSS1 expression restores the nuclear localization and HDR function of hypomorphic BRCA2 pathogenic mutants. ( A and B ) Nucleocytoplasmic distribution of the DBD mutants with DSS1 overexpression. HeLa cells expressing GFP-BRCA2 CT forms without (vector only) or with Myc-DSS1 were fixed with PFA and analysed by microscopy. Representative images are shown. Scale bar, 10μm. The graph (B) shows GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.) measured from the images. Myc-DSS1 expression increases the nuclear/cytoplasmic ratio of GFP-BRCA2 CT fragments to varying degrees: WT by 1.7 fold; W2626C and E2663V mutants by 3.5- to 5-fold; I2627F, R2659T, and G2748D mutants by 1.8- to 2.4-fold; T2722R, D2723H, and D2723G mutants, little change. More than 100 cells per sample were analysed. Statistical significance was assessed by one-way ANOVA test, followed by Bonferroni's multiple comparison test. A representative result from three independent experiments is shown. ( C ) mClover Lamin A HDR assay. HeLa cells harbouring BRCA2 mutations (Figure ) were transfected with vector or Myc-DSS1 plasmids in addition to mClover Lamin A HDR assay plasmids. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was assessed by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Expressing, Over Expression, Plasmid Preparation, Microscopy, Comparison, Transfection

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: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance. Our results suggest a hypothetical model wherein the engagement of DSS1 by the DBD of BRCA2 antagonizes BRCA2 self-oligomerization and promotes protein folding. Assembly of a correctly folded BRCA2/DSS1 complex enables nuclear transport. Cancer-causing mutations in the BRCA2 DBD impair DSS1 binding, perturbing this protein assembly mechanism, and provoking the formation of intracellular BRCA2 oligomers that are excluded from the cell nucleus. The failure to assemble correctly folded mutant BRCA2/DSS1 complexes, and cytosolic mis-localization of the mutant BRCA2, disables HDR in cells harbouring the DBD mutations. DSS1 can antagonize the self-oligomerization of wild-type BRCA2, and of cancer-causing DBD mutants such as W2626C that retain some degree of DSS1 binding.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Binding Assay, Mutagenesis