BRCA1–BARD1 promotes RAD51-mediated homologous DNA pairing (original) (raw)

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Acknowledgements

We thank X. Yu, J. Parvin and G. Dellaire for providing materials. This work was supported by US National Institutes of Health grants ES007061, CA220123, CA168635, CA92584, ES021454, CA215990 and R35GM118026. J.B.S. was supported by an NIH fellowship (F31CA210663). W.Z. and P.S. were also supported by a Basser Innovation Award from the Basser Center for BRCA at Penn Medicine’s Abramson Cancer Center.

Author information

Author notes

  1. Chen Sheng
    Present address: West China Hospital, Sichuan University, Chengdu, 610041, China

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, 06520, Connecticut, USA
    Weixing Zhao, Fengshan Liang, Youngho Kwon, Timsi Rao, Weibin Wang, Chen Sheng, Lucy Lu, Yong Xiong & Patrick Sung
  2. Department of Biochemistry & Molecular Biophysics, Columbia University, New York, 10032, New York, USA
    Justin B. Steinfeld, Chu Jian Ma & Eric C. Greene
  3. Department of Pediatrics, Section of Hematology-Oncology, Yale University School of Medicine, New Haven, 06520, Connecticut, USA
    Fengshan Liang, Xiaoyong Chen & Gary M. Kupfer
  4. Department of Pathology, Yale University School of Medicine, New Haven, 06520, Connecticut, USA
    Fengshan Liang, Xiaoyong Chen & Gary M. Kupfer
  5. Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, 80523, Colorado, USA
    David G. Maranon & Claudia Wiese
  6. Yale Center for Analytical Sciences, Yale School of Public Health, New Haven, Connecticut, USA
    Xuemei Song & Yanhong Deng
  7. Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, 06520, Connecticut, USA
    Judit Jimenez-Sainz, Ryan B. Jensen & Patrick Sung

Authors

  1. Weixing Zhao
  2. Justin B. Steinfeld
  3. Fengshan Liang
  4. Xiaoyong Chen
  5. David G. Maranon
  6. Chu Jian Ma
  7. Youngho Kwon
  8. Timsi Rao
  9. Weibin Wang
  10. Chen Sheng
  11. Xuemei Song
  12. Yanhong Deng
  13. Judit Jimenez-Sainz
  14. Lucy Lu
  15. Ryan B. Jensen
  16. Yong Xiong
  17. Gary M. Kupfer
  18. Claudia Wiese
  19. Eric C. Greene
  20. Patrick Sung

Contributions

W.Z. and P.S. conceived the study. W.Z., E.C.G., Y.X., G.M.K., C.W., and P.S. designed the experiments and analysed the data. W.Z., F.L., X.C., J.B.S., D.G.M., Y.K., C.J.M., T.R., W.W., C.S., L.L., J.J.-S. and R.B.J. generated key materials and executed the experiments. X.S. and Y.D. provided statistical analysis. W.Z. and P.S. wrote the paper with input from the other authors.

Corresponding authors

Correspondence toWeixing Zhao, Eric C. Greene or Patrick Sung.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks P. Cejka, R. Scully and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Purification of BRCA1–BARD1 and mutant variants, and DNA binding properties of BRCA1–BARD1 and BRCA1–BARD11–142.

a, b, Schematics of BRCA1 (a) and BARD1 (b) and mutant variants of these proteins tested in this study. c, SDS–PAGE of purified BRCA1–BARD11–142 (lane 2), BRCA1–BARD1 (lane 3), BRCA1–BARD1AAE (lane 4), BRCA11–500–BARD1 (lane 5), BRCA11–500–BARD11–261 (lane 6) and BRCA11–500–BARD1∆163–261 (lane 7). Size markers were run in lane 1. d, SDS–PAGE of purified BRCA1–BARD1 (lane 2), BRCA1–BARD1∆123–162 (lane 3), BRCA1–BARD1K140N (lane 4) and BRCA1∆758–1064–BARD1 (lane 5). Size markers were run in lane 1. e, DNA binding test of BRCA1–BARD1 with a mixture of D-loop, DNA bubble and dsDNA. f, Quantification of data from experiments in e. Data are means ± s.d., n = 5. g, DNA binding test of BRCA1–BARD1 with a mixture of D-loop, dsDNA and ssDNA. h, Quantification of data from experiments in g. Data are means ± s.d., n = 4. i, DNA binding test of BRCA1–BARD11–142 with a mixture of D-loop, DNA bubble and dsDNA. j, Quantification of the results obtained with 32 nM of protein complexes in e and i. Data are means ± s.d., n = 3 (BRCA1–BARD11–142) or 5 (BRCA1–BARD1). **P < 0.01.

Extended Data Figure 2 DNA binding by BARD1.

a, BRCA1–BARD1 (5 nM) was incubated with radiolabelled D-loop (10 nM) and then the nucleoprotein complex was presented with an increasing concentration of unlabelled ssDNA, dsDNA, fork, bubble or D-loop as indicated. b, Quantification of data from experiments in a. Data are means ± s.d., n = 2 (ssDNA) or 3 (all other substrates). c, DNA binding test of BRCA11–500–BARD11–261 with a mixture of D-loop, dsDNA and ssDNA. d, DNA binding test of BRCA11–500–BARD1∆163–261 with a mixture of D-loop, dsDNA and ssDNA. e, Comparison of results obtained using 32 nM of BRCA1–BARD1 (from Extended Data Fig. 1g), BRCA11–500–BARD1 (from Extended Data Fig. 10a), BRCA11–500–BARD11–261 (from c) and BRCA11–500–BARD1∆163–261 (from d). Data are means ± s.d., n = 3 (BRCA11–500–BARD11–261 and BRCA11–500–BARD1∆163–261) or 4 (BRCA1–BARD1 and BRCA11–500–BARD1). **P < 0.01. f, SDS–PAGE of purified BARD1124–270. g, EMSA to test BARD1124–270 for binding to the D-loop, DNA bubble (Bubble), double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). h, Nucleoprotein complex consisting of BARD1124–270 (16 nM) and radiolabelled D-loop (10 nM) was challenged with an increasing concentration of unlabelled ssDNA, dsDNA, fork, DNA bubble or D-loop as indicated. i, Quantification of data from experiments in h. Data are means ± s.d., n = 3 (D-loop and ssDNA) or 4 (Bubble, RF and dsDNA).

Source data

Extended Data Figure 3 The RAD51 interaction attributes of BRCA1–BARD1.

a, Affinity pull-down to test for the interaction of RecA with BRCA1–BARD1 (B1–B1) via the Flag tag on BRCA1. The supernatant (S), wash (W) and eluate (E) fractions were analysed by SDS–PAGE and Coomassie blue staining. b, Affinity pull-down with Flag-tagged BRCA1–BARD1 (66 nM) and an increasing concentration of RAD51 (1, 2, 4 and 8 μM). The eluates from the pull-down experiment were analysed by SDS–PAGE with Coomassie blue staining. c, The amount of BRCA1–BARD1 and RAD51 in lanes 2–5 of b was quantified against known quantities of these protein species, run and stained in the same SDS polyacrylamide gel. Data are means ± s.d., n = 3. d, Affinity pull-down to test for the interaction of RAD51 with BRCA1–BARD1 with or without ethidium bromide (EB) being present. e, Far western analysis to examine RAD51D–XRCC2 (DX2), GST–DSS1 (DSS1) and BRCA1–BARD1 for RAD51 interaction. f, Schematic of the GST-tagged RAD51 fragments examined (top). Results from the pull-down experiment to test for interaction of BRCA1-BARD1 with the RAD51 fragments via the GST tag on the latter (bottom). RAD51 fragments and BRCA1 were revealed by immunoblot analysis using anti-GST or anti-Flag antibodies, respectively. g, GST pull-down assay to test for the interaction of the RAD51-T3 fragment with BRCA1–BARD1, BRCA11–500–BARD1 and BRCA1–BARD11–142. The RAD51 fragment, GST, BRCA1 and BARD1 were revealed by immunoblot analysis using anti-GST, anti-Flag or anti-His antibodies, respectively. h, GST pull-down assay to test for competition between BRCA1–BARD1 (198 nM) and BRCA2–DSS1 (66 nM) for RAD51 (1 μM); DSS1 was GST-tagged. RAD51, BRCA1 and BRCA2 were revealed by immunoblot analysis using antibodies specific for them.

Extended Data Figure 4 Lack of recombination mediator activity in BRCA1–BARD1 and species-specific enhancement of RAD51 recombinase by BRCA1–BARD1.

a, Schematic of the test for mediator activity of BRCA complex (BRCA1–BARD1 and BRCA2–DSS1). b, BRCA1–BARD1 and BRCA2–DSS1 were tested for recombination mediator activity with RPA-coated ssDNA as substrate. c, Quantification of data from experiments in b. Data are means ± s.d., n = 3. d, Schematic of the test for ssDNA targeting activity of BRCA complex (BRCA1–BARD1 and BRCA2–DSS1). e, BRCA1–BARD1 was tested alongside BRCA2–DSS1 for the ability to target RAD51 to ssDNA. f, Quantification of data from experiments in e. Data are means ± s.d., n = 3. g, Schematic of the D-loop assay. h, D-loop reactions were carried out with the indicated concentration of BRCA1–BARD1 and ATP as the nucleotide cofactor. i, Quantification of data from experiments in h. Data are means ± s.d., n = 3. j, BRCA1–BARD1 and Saccharomyces cerevisiae Rad54 (yRad54) were tested for their influence on D-loop formation catalysed by S. cerevisiae Rad51 (yRad51). k, Quantification of data from experiments in j. Data are means ± s.d., n = 3.

Extended Data Figure 5 Interplay between BRCA2–DSS1 and BRCA1–BARD1.

a, D-loop reactions performed with the indicated concentration of BRCA1–BARD1 (B1–B1), BRCA2–DSS1 (B2–D1), and order of addition of reaction components. b, Quantification of data from experiments in a. Data are means ± s.d., n = 3. NS, non-significant. c, D-loop reactions performed with the indicated concentration of BRCA1–BARD1, BRCA2–DSS1, and order of addition of reaction components. d, Quantification of data from experiments in c. Data are means ± s.d., n = 3. *P < 0.05; **P < 0.01. e, Pairwise distance distributions39 for Atto565-dsDNA bound to the RAD51–ssDNA filaments with or without BRCA1–BARD1. Data are means ± errors (determined by bootstrapping). f, BRCA1–BARD1 (100 and 200 nM) was tested with filaments of yRad51–ssDNA in synaptic complex assembly as assayed by protection against restriction digest. g, Number of dsDNA oligonucleotides bound by the RAD51–ssDNA filament without (n = 49) and with BRCA1–BARD1 (n = 54), BRCA1–BARD1AAE (n = 50) or BRCA11–500–BARD1 (n = 50). Data are means ± 95% confidence intervals. **P < 0.01.

Source data

Extended Data Figure 6 Identification of the RAD51 interaction domain in BRCA1–BARD1.

a, Schematic of the BRCA1 deletion variants37 examined in this study. b, Testing BRCA1 deletion variants alone or in complex with BARD1 for the ability to co-immunoprecipitate RAD51 from insect cell extracts using anti-Flag resin with Benzonase treatment. The immunoprecipitates were analysed by western blotting with antibodies against the Flag epitope (for BRCA1), the His6 epitope (for BARD1), or RAD51, as indicated. The cell extracts (10% of total) were probed for their RAD51 content. c, Quantification of data from experiments in b. Data are means ± s.d., n = 3. *P < 0.05; **P < 0.01. d, Summary of the RAD51 interaction ability of BARD1 truncation mutants, based on the pull-down analyses in e (for BRCA1–BARD1, BRCA11–500–BARD1 and BRCA11–500–BARD11–261), f (for BRCA11–500–BARD1, BRCA11–500–BARD11–261 and BRCA11–500–BARD11–122), g (for BRCA11–500–BARD1∆123–261, BRCA11–500–BARD1∆123–162, BRCA11–500–BARD11–261 and BRCA11–500–BARD11–162) and h (for BARD1123–162). In e, f and g, the eluates from the affinity resin were analysed by SDS–PAGE and Coomassie blue staining. In h, the interaction between RAD51 and GST–BARD1123–162 was tested by pull-down using glutathione resin. The input and eluate fractions were analysed by western blotting with antibodies against GST or RAD51, as indicated.

Extended Data Figure 7 Characterization of BRCA1–BARD1 mutants.

a, BRCA1–BARD1 (n = 3), BRCA1–BARD1AAE (n = 3), BRCA1–BARD1∆123–162 (n = 3), and BRCA1–BARD1K140N (n = 4) were tested for their DNA binding activity using a mixture of radiolabelled D-loop and dsDNA as substrates. b, Quantification of data from experiments in a. Data are means ± s.d. c, Wild-type and mutant variants of BRCA1–BARD1 (300 nM each) were tested for the ability to promote synaptic complex formation. d, Quantification of data from experiments in c. Data are means ± s.d., n = 3. e, Synaptic complex formation by RAD51–ssDNA filament with BRCA1–BARD1 (100 and 200 nM) and BRCA1∆758–1064–BARD1 (100 and 200 nM). f, Quantification of data from experiments in e. Data are means ± s.d., n = 6 (BRCA1–BARD1 with Mg2+ and ATP) or n = 2 (all other conditions). *P < 0.05; **P < 0.01.

Extended Data Figure 8 Role of BRCA1 and BARD1 in homologous recombination and RAD51 focus formation.

a, Western blot to verify the nuclear localization of endogenous BRCA1 and ectopically expressed Flag-SBP-tagged BARD1 or the AAE mutant in HeLa cells. The cytoplasmic (C) and nuclear (N) fractions were also analysed for their alpha-tubulin and histone H3 contents. b, Western blot analysis to detect endogenous BRCA1 and BARD1 after treatment of DR-U2OS cells with BRCA1 or BARD1 siRNA. c, Homologous recombination frequency in DR-U2OS cells with siRNA-mediated knockdown of BRCA1 or BARD1. Data are means ± s.d., n = 3. d, Gene-targeting efficiency of CRISPR–CAS9 in U2OS cells with siRNA-mediated knockdown of BRCA1 or BARD1. Data are means ± s.d., n = 3. e, Western blot analysis to detect endogenous BRCA1, BARD1 and BRCA2 after treatment of HeLa cells with siRNA against BRCA1, BARD1 or BRCA2. Alpha-tubulin serves as loading control. f, Representative micrographs of RAD51 foci (red) in the nuclei of HeLa cells treated with BRCA1, BARD1, BRCA2 or control siRNA 8 h after exposure to 4 Gy γ-rays. Blue, DAPI. g, Quantification of RAD51 foci at various time points after exposure to 4 Gy γ-rays or sham irradiation. The mean values ± s.e.m. of 4 (siBRCA2 and siBARD1), 6 (siBRCA1) or 7 (siControl) independent experiments are shown. h, Western blot analysis to detect endogenous BRCA1 and 53BP1 after treatment of DR-U2OS cells with BRCA1 or TP53BP1 siRNA. i, Homologous recombination frequency in DR-U2OS cells with siRNA-mediated knockdown of BRCA1 and/or TP53BP1. Data are means ± s.d., n = 3. j, Western blot analysis to detect endogenous BARD1 and 53BP1 after treatment of DR-U2OS cells with BARD1 and/or TP53BP1 siRNA. k, Homologous recombination frequency in DR-U2OS cells with siRNA-mediated knockdown of BARD1 or TP53BP1. Data are means ± s.d., n = 3. l, Western blot analysis to detect ectopically expressed and endogenous BARD1 after treatment of U2OS cells with BARD1 and/or TP53BP1 siRNA. As the abundance of ectopically expressed Flag-SBP-tagged wild-type and mutant BARD1 was lower than that of endogenous BARD1, we revealed it with anti-Flag antibodies in western blot analysis. m, Homologous recombination frequency in DR-U2OS cells treated with siRNA against BARD1 and/or TP53BP1 and stably expressing BARD1WT_res_ or BARD1AAE_res_. Data are means ± s.d., n = 3. *P < 0.05; **P < 0.01; NS, non-significant.

Source data

Extended Data Figure 9 Characterization of human cells expressing BARD1 mutants.

a, Western blot analysis to detect ectopically expressed and endogenous BARD1 after treatment of U2OS cells with BARD1 or control siRNA for the experiments in Fig. 5b. b, Western blot analysis to detect ectopically expressed and endogenous BARD1 after treatment of U2OS cells with BARD1 or control siRNA for the experiments in Fig. 5c. c, Western blot analysis to detect ectopically expressed and endogenous BARD1 after treatment of HeLa cells with BARD1 or control siRNA for the experiments in Fig. 5d. In a–c, as the abundance of ectopically expressed Flag-SBP-tagged wild-type and mutant BARD1 was lower than that of endogenous BARD1, we revealed it with anti-Flag antibodies in western blot analysis. d, Representative micrographs of RAD51 foci (red) in the nuclei of HeLa cells expressing Flag-SBP-tagged BARD1WT_res_ or BARD1AAE_res_ 8 h after exposure to 4 Gy γ-rays. Blue, DAPI. e, Quantification of RAD51 foci at various time points after exposure to 4 Gy γ-rays or sham irradiation. The mean values ± s.e.m. of 5 (8-h time point) or 3 (all other time points) independent experiments are shown. NS, non-significant. f, Western blot to reveal pRPA32(S4/S8) (with tubulin as the loading control) at various time points (0, 24 and 72 h) after a 1-h treatment with 2 μM MMC.

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Extended Data Figure 10 Characterization of BRCA11–500–BARD1 and BRCA1∆758–1064–BARD1.

a, BRCA11–500–BARD1 was tested for DNA binding using a mixture of radiolabelled D-loop, dsDNA, and ssDNA as substrates. b, Quantification of data from experiments in a. Data are means ± s.d., n = 4. c, Comparison of results obtained using 32 nM BRCA1–BARD1 (from Extended Data Fig. 1g) and BRCA11–500–BARD1 (from a). Data are means ± s.d., n = 3. NS, non-significant. d, BRCA1–BARD1 and BRCA1∆758–1064–BARD1 were tested for DNA binding using a mixture of radiolabelled D-loop, bubble, and dsDNA as substrates. e, Comparison of results obtained using 16 nM BRCA1–BARD1 and BRCA1∆758–1064–BARD1. Data are means ± s.d., n = 4. NS, non-significant. f, Far western analysis to detect RAD51 association with BRCA11–500 and BARD1 immobilized on nitrocellulose membrane. g, Pull-down assay to test for the interaction of RAD51 with BRCA11–500–BARD1, BRCA1–BARD11–142 and BRCA1–BARD1 via the Flag tag on the BRCA1 species. The eluates from the various anti-Flag resin fractions were subjected to immunoblot analysis with anti-Flag (for BRCA1), anti-His (for BARD1) and anti-RAD51 antibodies. h, Pull-down assay to test for the interaction between RAD51 and BRCA1–BARD1 or BRCA1∆758–1064–BARD1 via the Flag tag on the BRCA1 species. i, BRCA11–500–BARD1 and BRCA1∆758–1064–BARD1 were tested along with the wild-type complex for the ability to enhance RAD51-mediated D-loop formation. j, Quantification of data from experiments in i. Data are means ± s.d., n = 3 or 4. **P < 0.01.

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Zhao, W., Steinfeld, J., Liang, F. et al. BRCA1–BARD1 promotes RAD51-mediated homologous DNA pairing.Nature 550, 360–365 (2017). https://doi.org/10.1038/nature24060

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