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Isomerization of BRCA1–BARD1 promotes replication fork protection

Nature volume 571, pages 521–527 (2019)Cite this article

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Abstract

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1–PALB2 interaction, is required for fork protection. BRCA1–BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1–BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1–BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1–BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.

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Fig. 1: BRCA1 phosphorylation at Ser114 is required for fork protection.
Fig. 2: PIN1 regulates BRCA1–BARD1 in fork protection.
Fig. 3: BRCA1 isomerization enhances RAD51 binding.
Fig. 4: Loss of isomerization of BRCA1 leads to genomic instability.
Fig. 5: Constitutively expressed BRCA1(P115A) is genotoxic.
Fig. 6: Loss of fork protection in BRCA1–BARD1 patient variants.

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Data availability

All datasets that were generated during the current study are provided as online source data associated with this paper. The custom mouse monoclonal (3C10G8) and rabbit polyclonal antibodies that were raised against BRCA1(pS114) are available on request to the corresponding authors subject to completion of a standard MTA.

References

  1. Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Sidorova, J. A game of substrates: replication fork remodeling and its roles in genome stability and chemo-resistance. Cell Stress 1, 115–133 (2017).

    PubMed  PubMed Central  Google Scholar 

  3. Cantor, S. B. & Calvo, J. A. Fork protection and therapy resistance in hereditary breast cancer. Cold Spring Harb. Symp. Quant. Biol. 82, 339–348 (2017).

    PubMed  Google Scholar 

  4. Hashimoto, Y., Ray Chaudhuri, A., Lopes, M. & Costanzo, V. Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat. Struct. Mol. Biol. 17, 1305–1311 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Schlacher, K. et al. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145, 529–542 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Zadorozhny, K. et al. Fanconi-anemia-associated mutations destabilize RAD51 filaments and impair replication fork protection. Cell Rep. 21, 333–340 (2017).

    CAS  PubMed  Google Scholar 

  7. Wang, A. T. et al. A dominant mutation in human RAD51 reveals its function in DNA interstrand crosslink repair independent of homologous recombination. Mol. Cell 59, 478–490 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Ameziane, N. et al. A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51. Nat. Commun. 6, 8829 (2015).

    ADS  CAS  PubMed  Google Scholar 

  9. Higgs, M. R. & Stewart, G. S. Protection or resection: BOD1L as a novel replication fork protection factor. Nucleus 7, 34–40 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Dungrawala, H. et al. RADX promotes genome stability and modulates chemosensitivity by regulating RAD51 at replication forks. Mol. Cell 67, 374–386 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bhat, K. P. et al. RADX modulates RAD51 activity to control replication fork protection. Cell Rep. 24, 538–545 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ray Chaudhuri, A. et al. Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387 (2016).

    ADS  PubMed  Google Scholar 

  13. Yazinski, S. A. et al. ATR inhibition disrupts rewired homologous recombination and fork protection pathways in PARP inhibitor-resistant BRCA-deficient cancer cells. Genes Dev. 31, 318–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Feng, W. & Jasin, M. BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination. Nat. Commun. 8, 525 (2017).

    ADS  PubMed  PubMed Central  Google Scholar 

  15. Dungrawala, H. & Cortez, D. Purification of proteins on newly synthesized DNA using iPOND. Methods Mol. Biol. 1228, 123–131 (2015).

    PubMed  PubMed Central  Google Scholar 

  16. Sirbu, B. M. et al. Identification of proteins at active, stalled, and collapsed replication forks using isolation of proteins on nascent DNA (iPOND) coupled with mass spectrometry. J. Biol. Chem. 288, 31458–31467 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Schlacher, K., Wu, H. & Jasin, M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell 22, 106–116 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang, F., Fan, Q., Ren, K. & Andreassen, P. R. PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2. Mol. Cancer Res 7, 1110–1118 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang, F. et al. PALB2 links BRCA1 and BRCA2 in the DNA-damage response. Curr. Biol. 19, 524–529 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Sy, S. M., Huen, M. S. & Chen, J. PALB2 is an integral component of the BRCA complex required for homologous recombination repair. Proc. Natl Acad. Sci. USA 106, 7155–7160 (2009).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhao, W. et al. BRCA1–BARD1 promotes RAD51-mediated homologous DNA pairing. Nature 550, 360–365 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Paull, T. T., Cortez, D., Bowers, B., Elledge, S. J. & Gellert, M. Direct DNA binding by Brca1. Proc. Natl Acad. Sci. USA 98, 6086–6091 (2001).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Densham, R. M. et al. Human BRCA1–BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection. Nat. Struct. Mol. Biol. 23, 647–655 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Hayami, R. et al. Down-regulation of BRCA1–BARD1 ubiquitin ligase by CDK2. Cancer Res. 65, 6–10 (2005).

    CAS  PubMed  Google Scholar 

  25. Mertins, P. et al. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat. Methods 10, 634–637 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Steger, M. et al. Prolyl isomerase PIN1 regulates DNA double-strand break repair by counteracting DNA end resection. Mol. Cell 50, 333–343 (2013).

    CAS  PubMed  Google Scholar 

  27. Zheng, H. et al. The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response. Nature 419, 849–853 (2002).

    ADS  CAS  PubMed  Google Scholar 

  28. Nepomuceno, T. C. et al. BRCA1 recruitment to damaged DNA sites is dependent on CDK9. Cell Cycle 16, 665–672 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Weiss, M. S., Jabs, A. & Hilgenfeld, R. Peptide bonds revisited. Nat. Struct. Biol. 5, 676 (1998).

    CAS  PubMed  Google Scholar 

  30. Alderson, T. R., Lee, J. H., Charlier, C., Ying, J. & Bax, A. Propensity for cis-proline formation in unfolded proteins. ChemBioChem 19, 37–42 (2018).

    PubMed  Google Scholar 

  31. Göthel, S. F. & Marahiel, M. A. Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts. Cell. Mol. Life Sci. 55, 423–436 (1999).

    PubMed  Google Scholar 

  32. Ranganathan, R., Lu, K. P., Hunter, T. & Noel, J. P. Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell 89, 875–886 (1997).

    CAS  PubMed  Google Scholar 

  33. Lu, K. P., Hanes, S. D. & Hunter, T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 380, 544–547 (1996).

    ADS  CAS  PubMed  Google Scholar 

  34. Yaffe, M. B. et al. Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science 278, 1957–1960 (1997).

    ADS  CAS  PubMed  Google Scholar 

  35. Lu, P. J., Zhou, X. Z., Shen, M. & Lu, K. P. Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 283, 1325–1328 (1999).

    ADS  CAS  PubMed  Google Scholar 

  36. Nakamura, K. et al. Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease. Cell 149, 232–244 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Hilton, B. A. et al. ATR plays a direct antiapoptotic role at mitochondria, which is regulated by prolyl isomerase Pin1. Mol. Cell 60, 35–46 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhou, X. Z. et al. Pin1-dependent prolyl isomerization regulates dephosphorylation of Cdc25C and tau proteins. Mol. Cell 6, 873–883 (2000).

    CAS  PubMed  Google Scholar 

  39. Innes, B. T., Bailey, M. L., Brandl, C. J., Shilton, B. H. & Litchfield, D. W. Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding. Front. Physiol. 4, 18 (2013).

    PubMed  PubMed Central  Google Scholar 

  40. Taglialatela, A. et al. Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers. Mol. Cell 68, 414–430 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Petermann, E., Orta, M. L., Issaeva, N., Schultz, N. & Helleday, T. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol. Cell 37, 492–502 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Hanada, K. et al. The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat. Struct. Mol. Biol. 14, 1096–1104 (2007).

    CAS  PubMed  Google Scholar 

  43. Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).

    PubMed  Google Scholar 

  44. Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013).

    PubMed  PubMed Central  Google Scholar 

  45. Hedau, S. et al. Novel germline mutations in breast cancer susceptibility genes BRCA1, BRCA2 and p53 gene in breast cancer patients from India. Breast Cancer Res. Treat. 88, 177–186 (2004).

    CAS  PubMed  Google Scholar 

  46. Chapman, J. R., Taylor, M. R. & Boulton, S. J. Playing the end game: DNA double-strand break repair pathway choice. Mol. Cell 47, 497–510 (2012).

    CAS  PubMed  Google Scholar 

  47. Min, S. H. et al. Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase. Mol. Cell 46, 771–783 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Lu, K. P., Finn, G., Lee, T. H. & Nicholson, L. K. Prolyl cis-trans isomerization as a molecular timer. Nat. Chem. Biol. 3, 619–629 (2007).

    CAS  PubMed  Google Scholar 

  49. Billing, D. et al. The BRCT domains of the BRCA1 and BARD1 tumor suppressors differentially regulate homology-directed repair and stalled fork protection. Mol. Cell 72, 127–139 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Ding, X. et al. Synthetic viability by BRCA2 and PARP1/ARTD1 deficiencies. Nat. Commun. 7, 12425 (2016).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  51. Berger, I., Fitzgerald, D. J. & Richmond, T. J. Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22, 1583–1587 (2004).

    CAS  PubMed  Google Scholar 

  52. Subramanyam, S., Ismail, M., Bhattacharya, I. & Spies, M. Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics. Proc. Natl Acad. Sci. USA 113, E6045–E6054 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529 (2017).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Grant funding was as follows. CRUK: C8820/A19062 (R.M.D., G.E.R., K.S., H.L.M., H.R.S., J.F.J.B., J.L.C.), C17183/A23303 (G.S.S.); Breast Cancer Now: 2015MayPR499 (K.S.); Wellcome Trust: 206343/Z/17/Z (M.J., A.K.W., A.J.G., A.S.C.), 210658/Z/18/Z (X.Z.); MRC: MR/N000188/1 (X.Z.); and as part of the CRUK centre training (M.D.-M.).We thank S. Cook for the pGEX4T1-PIN1 plasmid, T. Sixma for the BARD127–327 bacterial expression construct, J. Stark for U20S DR-GFP cells, Y. Sun for recombinant RAD51, P. Byrd for advice regarding PALB2 reagents, T. Wallach for generating the S114P mutation, J. Reynolds for advice on metaphase spreads and S. Begum for support with PLAs. In addition, we thank the Microscopy and Imaging Services (MISBU) and the Flow Cytometry Services (UoBFC) in the Tech Hub facility at Birmingham University for support and maintenance of microscopes and FACS equipment.

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Author notes

  1. Manuel Daza-Martin

    Present address: The Institute of Cancer Research, Chester Beatty Laboratories, London, UK

  2. Jennifer L. Coles

    Present address: Warwick Medical School, The University of Warwick, Coventry, UK

Authors and Affiliations

  1. Birmingham Centre for Genome Biology and Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK

    Manuel Daza-Martin, Katarzyna Starowicz, Mohammed Jamshad, George E. Ronson, Hannah L. MacKay, Anoop Singh Chauhan, Alexandra K. Walker, Helen R. Stone, James F. J. Beesley, Jennifer L. Coles, Alexander J. Garvin, Grant S. Stewart, Ruth M. Densham & Joanna R. Morris

  2. Section of Structural Biology, Department of Medicine, Imperial College London, London, UK

    Stephanie Tye, Thomas J. McCorvie & Xiaodong Zhang

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Contributions

M.D.-M. generated reagents, performed survival, PLA and fibre experiments and in vitro analysis. R.M.D. generated reagents, performed fibre experiments, in vitro RAD51 assays and survival analysis. K.S. performed metaphase experiments and long-term analysis of the P115A mutation. M.J. generated purified proteins and performed in vitro analysis. T.J.M. generated full-length BRCA1–BARD1 and performed initial analysis. S.T. purified full-length BRCA1–BARD1 and performed in vitro RAD51 binding and trypsin digestion. G.E.R. performed western blotting and Flag immuoprecipitation. A.S.C. performed GST–WW analyses. H.L.M. performed the EdU–BRCA1(pS114) assay. A.K.W. analysed RAD51 foci (BRCA1 variants). H.R.S. analysed RAD51 foci (BARD1 variants). J.F.J.B. provided technical support. J.L.C. generated BRCA1(M1411T) cells and performed cisplatin survival analysis. A.J.G. generated constructs and cell lines. G.S.S. made preliminary observations. X.Z. supervised S.T. and T.J.M. J.R.M., R.M.D. and M.D.-M. wrote the paper. All authors commented on the paper and research. R.M.D and J.R.M conceived and directed the project.

Corresponding authors

Correspondence to Ruth M. Densham or Joanna R. Morris.

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Extended data figures and tables

Extended Data Fig. 1 The canonical BRCA1–PALB2 interaction is not required for fork protection.

a, Schematic of the DNA fibre assay that was used to measure fork protection by calculating IdU:CldU ratios. b, c, IdU:CldU ratios from U20S cells in which BRCA1 or PALB2 expression was knocked down by siRNA transfection and cells were treated with hydroxyurea (5 mM, 3 h). n = 300 fibres from 3 biological replicates; bars depict median ± 95% CI. c, Representative blot (n = 3). d, Schematic of BRCA1 protein, indicating RING (red), RAD51-binding (green), coiled-coil (blue) and BRCT repeat (purple) domains. The M1411T patient variant disrupts binding of PALB2 and is located in the coiled-coil domain. e, Schematic of PALB2 protein, indicating BRCA1-interacting coiled-coil (blue), ChAM and DNA-binding (purple and green) and WD40-like repeat (orange) domains. The PALB2(ΔNT) mutant lacks the N-terminal coiled-coil domain. f, Colony survival following cisplatin treatment (2.5 µM, 2 h) in HeLa cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(M1411T). n = 4; data are mean ± s.e.m. g, Colony survival after cisplatin treatment (2 h) in U20S cells in which PALB2 expression was knocked down by siRNA transfection and cells were complemented with Flag-tagged wild-type PALB2 or Flag-tagged PALB2(ΔNT). n = 4; data are mean ± s.e.m. h, Representative blot for j (n = 3). i, Representative blot for k (n = 3). j, IdU:CldU ratios from U20S cells in which BRCA1 expression was knocked down by siRNA transfection, cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(M1411T) and treated with hydroxyurea (5 mM, 3 h). n = 300 fibres from 3 biological replicates; bars depict median ± 95% CI. k, IdU:CldU ratios from U20S cells in which PALB2 expression was knocked down by siRNA transfection, cells were complemented with Flag-tagged wild-type PALB2 or Flag-tagged PALB2(ΔNT) and treated with hydroxyurea (5 mM, 3 h). n = 300 fibres from 3 biological replicates; bars depict median ± 95% CI. l, Percentage of stalled replication forks in U20S cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(M1411T). n = 3; data are mean ± s.e.m. m, As for l but for knockdown of PALB2. Cells were complemented with Flag-tagged wild-type PALB2 (n = 3) or Flag-tagged PALB2(ΔNT) (n = 3). n = 2 for NTC and siPALB2; data are mean ± s.e.m. n, Percentage of replication forks that were able to restart after hydroxyurea treatment (5 mM, 3 h) in U20S cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(M1411T). n = 3; data are mean ± s.e.m. o, As for n but for knockdown of PALB2. Cells were complemented with Flag-tagged wild-type PALB2 (n = 3) or Flag-tagged PALB2(ΔNT) (n = 3). n = 2 for NTC and siPALB2; data are mean ± s.e.m. A two-sided unpaired t-test was used to calculate all P values.

Source Data

Extended Data Fig. 2 BRCA1–BARD1-mediated fork protection requires the RAD51-binding domain of BARD1.

a, b, Colony survival after 2-h treatment with olaparib (a; n = 4) or 16-h treatment with hydroxyurea (b; n = 7) in U20S cells in which BARD1 expression was knocked down by siRNA transfection and cells were complemented with RFP–Flag-tagged wild-type BARD1 or RFP–Flag-tagged BARD1(AAE). Data are mean ± s.e.m. c, d, IdU:CldU ratios from U20S cells in which BARD1 expression was knocked down by siRNA transfection, cells were complemented with RFP–Flag-tagged wild-type BARD1 or RFP–Flag-tagged BARD1(AAE) and treated with hydroxyurea (5 mM, 3 h). n = 315 fibres from 3 biological replicates; bars depict median ± 95% CI. d, Representative blot (n = 3). e, f, As for c, but cells were complemented with RFP–Flag-tagged wild-type BARD1 or RFP–Flag-tagged BARD1(R99E). n = 300 fibres from 3 biological replicates; bars depict median ± 95% CI. f, Representative blot (n = 3). A two-sided unpaired t-test was used to calculate all P values.

Source Data

Extended Data Fig. 3 The role of BRCA1–BARD1 phosphorylation sites that are proximal to RING domains in replication stress.

a–e, IdU:CldU ratios (a) and CldU track lengths (b, d) from U20S cells in which BARD1 expression was knocked down by siRNA transfection, cells were complemented with RFP–Flag-tagged BARD1 variants and treated with hydroxyurea (5 mM, 3 h). n = 300 and n = 600 fibres from 3 biological replicates (a and b, respectively), or n = 290 fibres from 2 biological replicates (d); bars depict median ± 95% CI. c, e, Representative blots (n = 3 (c) and n = 2 (e)). f, The percentage of foci in which BRCA1 and CldU were co-localized per cell was calculated for U20S cells expressing Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(S114A). n = 25 cells; bars depict median ± 95% CI. g, Representative blot of Flag immunoprecipitation of Flag–eGFP-tagged BRCA1 variants from HEK293 cells (n = 3). h, i, Percentage of stalled replication forks (h) and percentage of replication forks that were able to restart (i) after hydroxyurea treatment (5 mM, 3 h) in U20S cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(S114A). n = 3; data are mean ± s.e.m. j, k, As for b, but for knockdown of BRCA1. Cells were complemented with Flag–eGFP-tagged BRCA1(S114A) and treated with mirin (50 μM) and hydroxyurea (5 mM) for 3 h. n = 245 fibres from 3 biological replicates; bars depict median ± 95% CI. k, Representative blot (n = 3). A two-sided unpaired t-test was used to calculate all P values.

Source Data

Extended Data Fig. 4 Phosphorylation of BRCA1 at Ser114 promotes PIN1 interaction.

a, Left, schematic of the protein structure of PIN1, indicating the WW domain (which binds to phosphorylated serine/threonine residues ahead of proline residues) and PPIase domain. The cartoons illustrate the recombinant constructs of GST fused to the wild-type WW domain or the WW(W34A) mutant. Right, Coomassie blue staining of recombinant GST–WW fragments purified from E. coli. b, c, Densitometry quantification (b) and representative blot (c; n = 3) of GST–WW pull-downs from U20S cells expressing Flag–eGFP-tagged wild-type BRCA1 and Flag–eGFP-tagged BRCA1(S114A). Beads that were bound by GST–WW(W34A) were used as a negative control. A representative image is shown in Fig. 1e. Four independent experiments were performed; data are mean ± s.e.m. A two-sided unpaired t-test was used to calculate all P values. d, As for c but for cells expressing Flag–eGFP-tagged wild-type BRCA1, with and without hydroxyurea treatment (5 mM, 3 h). Representative blot (n = 3). e, As for d but in HEK293 cells and probed for endogenous BRCA1. The final lane indicates lysates that were pre-treated with CIP. Representative blot (n = 3). f, As for c but in U20S cells expressing RFP–Flag-tagged wild-type BARD1 and RFP–Flag-tagged BARD1(S148A). Representative blot (n = 3). g, Table with details of the inhibitors of proline-directed kinases that were used in h. h, GST–WW pull-downs from HEK293 cells expressing Flag–eGFP-tagged BRCA1, treated with and without the kinase inhibitors described in g. Beads that were bound by GST–WW(W34A) were used as a negative control. Representative blot (n = 3). i, GST–WW pull downs from HEK293 cells expressing Flag–eGFP-tagged BRCA1 and in which CDK1 expression was knocked down by siRNA transfection. j, Coomassie blue staining of purified recombinant His-tagged wild-type BRCA11–300 or BRCA11–300 (S114A) in complex with His-tagged BARD126-142 (representative gel; n = 3). k, Purified recombinant His–BRCA11-300–His–BARD126-142 was incubated with recombinant active CDK1 and cyclin A2, CDK2 and cyclin A, or CDK9 and cyclin K. Western blots were probed for BRCA1(pS114) (using 3C10G8 antibody) and BRCA1 (representative blot; n = 3). l, Recombinant purified wild-type or BRCA1(S114A) His–BRCA11–300–His–BARD126–142 were incubated with recombinant active CDK2 and cyclin A. Western blots were probed for BRCA1(pS114) (3C10G8 antibody) and BRCA1 (representative blot; n = 3).

Source Data

Extended Data Fig. 5 PIN1 regulates the BRCA1–BARD1 heterodimer to promote fork protection.

a, Tract lengths of CldU fibres were measured from U20S cells in which BRCA1 expression was knocked down by siRNA transfection and cells were treated with hydroxyurea (5 mM) and juglone (20 μM) for 3 h. n = 450 fibres from 3 biological replicates; bars depict median ± 95% CI. b, c, Tract lengths of CldU fibres were measured from U20S cells in which BARD1 and/or PIN1 expression was knocked down by siRNA transfection and cells were treated with hydroxyurea (5 mM, 3 h). n = 300 fibres from 2 biological replicates; bars depict median ± 95% CI. c, Representative blot (n = 2). d, e, Representative blot (d) and quantification (e) of BRCA1 expression (normalized to β-actin levels) after knockdown of PIN1. n = 9; data are mean ± s.e.m. f, g, IdU:CldU ratios from U20S cells in which BRCA1 expression was knocked down by siRNA transfection, cells were complemented with Flag–eGFP-tagged BRCA1 variants and treated with hydroxyurea (5 mM, 3 h). n = 200 fibres from 2 biological replicates; bars depict median ± 95% CI. g, Representative blot (n = 2). A two-sided unpaired t-test was used to calculate all P values.

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Extended Data Fig. 6 BRCA1–BARD1 isomerization enhances binding of RAD51.

a, His-tagged wild-type BRCA11–500 or His-tagged BRCA11–500(S114D) and BARD127–327 were incubated with full-length GST–PIN1 to induce isomerization and incubated with recombinant active RAD51, and their ability to bind RAD51 was assessed by His purification of the complex followed by western blotting. Representative image (n = 2). b, Representative Coomassie-blue-stained gel of purified recombinant full-length wild-type GST–PIN1 and GST–PIN1(C113S) from E. coli (n = 2). c, Representative Coomassie-blue-stained gel of purified recombinant His-tagged BRCA11–500 and BARD127–327 from E. coli (n = 3). d, Recombinant RAD51 was incubated with recombinant full-length BRCA1–BARD1 and BRCA1(P115A)–BARD1 in the presence of ATP and Mg2+. The ability of BRCA1–BARD1 to bind RAD51 was assessed by Strep pull-down of BRCA1, followed by SDS–PAGE and staining with Coomassie blue (n = 2). e, Fold change in the amount of recombinant RAD51 bound to recombinant full-length BRCA1–BARD1 and BRCA1–BARD1(P115A) in the presence of ATP and Mg2+, relative to wild-type BRCA1–BARD1. n = 6 (2 biological replicates, each with 3 technical replicates); data are mean ± s.e.m. f, Quantification of RAD51, co-immunoprecipitated with Flag–eGFP-tagged BRCA1 and RFP–Flag-tagged BARD1 from HEK293 cells, normalized to the level of BRCA1–BARD1 precipitated. n = 12; data are mean ± s.e.m. g, Representative blot for f. h, Representative gel for Fig. 3c, d. Recombinant full-length BRCA1–BARD1 was incubated with trypsin, with samples taken at the times indicated. The limited proteolysis profiles were assessed by SDS–PAGE and staining with Coomassie blue (n = 3). i, Representative images for Fig. 3e. RAD51 co-localization with nascent DNA, marked by pulse labelling with EdU, was measured by PLA in U20S cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged BRCA1 variants as indicated. Red foci indicate interaction between RAD51 and EdU–biotin in cells. Scale bars, 10 µm. A two-sided unpaired t-test was used to calculate all P values.

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Extended Data Fig. 7 BRCA1(S114A) shows increased sensitivity to agents that induce replication stress.

a–d, Colony survival following treatment with aphidicolin (16 h) (a) or the PARP inhibitors olaparib (b), veliparib (c) and 4AN (d) (all 2 h) was measured in HeLa cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(S114A). The number of replicates (n) is shown in parentheses for each condition. Data are mean ± s.e.m. e, Colony survival following 16-h treatment with hydroxyurea was measured in U20S cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(S114A). n = 3; data are mean ± s.e.m. f, g, Formation of RAD51 foci was measured in S-phase U20S cells (labelled with EdU) in which BRCA1 expression was knocked down by siRNA transfection, cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(S114A) and treated with olaparib (20 µM, 2 h). f, Representative images. Scale bars, 10 μm. g, Quantification of the number of RAD51 foci per EdU-positive cell. The number of replicates (n) is shown in parentheses for each condition. Bars depict median ± 95% CI. h, U20S cells that were induced to constitutively express Flag–eGFP-tagged wild-type BRCA1 or Flag–eGFP-tagged BRCA1(P115A) were stained for γH2AX foci as a marker for the accumulation of DNA damage over time. The number of γH2AX foci was counted in EdU-negative cells. Data are combined from three biological replicates. n = 90 cells; data are mean ± s.e.m. A two-sided unpaired t-test was used to calculate all P values.

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Extended Data Fig. 8 Patient variants define a region of BRCA1 that is required for fork protection.

a, Colony survival following 2-h treatment with olaparib was measured in cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or BRCA1 variants (Y101N, Y179C, S184C, S265Y in U20S cells; S114P and R133C in HeLa cells). The number of replicates (n) is shown in parentheses for each condition. Data are mean ± s.e.m. b, c, As for a, but with continuous treatment with olaparib (b) or cisplatin (c), and all patient variants were expressed in U20S cells. d, e, Formation of RAD51 foci was measured 2 h after 2-Gy irradiation in U20S cells in which BRCA1 expression was knocked down by siRNA transfection and cells were complemented with Flag–eGFP-tagged wild-type BRCA1 or BRCA1 patient variants as indicated. d, Representative images for e. Scale bars, 10 µm. e, Quantification of the number of RAD51 foci per EdU-positive cell. n = 150 cells from 3 biological replicates; bars depict median ± 95% CI. A two-sided unpaired t-test was used to calculate all P values.

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Extended Data Fig. 9 Patient variants define a region of BARD1 that is required for fork protection.

a, Colony survival following 2-h treatment with olaparib was measured in U20S cells in which BARD1 expression was knocked down by siRNA transfection and cells were complemented with RFP–Flag-tagged wild-type BARD1 or BARD1 variants as indicated. The number of replicates (n) is shown in parentheses for each condition. Data are mean ± s.e.m. b, c, As for a, but with continuous treatment with olaparib (b) or cisplatin (c). d, e, Formation of RAD51 foci was measured 2 h after 2-Gy irradiation in U20S cells in which BARD1 expression was knocked down by siRNA transfection and cells were complemented with RFP–Flag-tagged wild-type BARD1 or BARD1 patient variants as indicated. d, Representative images for e. Scale bars, 10 µm. e, Quantification of the number of RAD51 foci per EdU-positive cell. Graphed data are combined from three biological replicates; bars depict median ± 95% CI. Actual n values for each condition are shown in parentheses. f, Homologous recombination (U2OS-DR3-GFP) assays in cells in which BARD1 expression was knocked down by siRNA transfection. Cells were transfected with an I-SceI-expression construct and either RFP or the RFP–Flag-tagged BARD1 variants indicated, and counted by FACS analysis. GFP-positive cells were normalized to RFP-positive cells as a measure of transfection efficiency. The percentage of homologous recombination repair is given relative to siRNA-transfected NTC. n = 6; data are mean ± s.e.m. The FACS gating strategy is described in Supplementary Fig. 1. A two-sided unpaired t-test was used to calculate all P values.

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Extended Data Fig. 10 Isomerization of phosphorylated BRCA1–BARD1 promotes replication fork protection.

a, Table summarizing the survival and replication fork stability responses to DNA-damaging agents by the variants of BRCA1–BARD1 that were used in this study. b, Model to illustrate CDK1 or CDK2 (grey) phosphorylation at Ser114 (red) and subsequent PIN1 (purple) isomerization events on BRCA1 (green) and BARD1 (orange). BRCA1 isomerization enhances the ability of BARD1 to associate with RAD51 (brown) and thereby promotes replication fork protection.

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Supplementary Information

This file contains Supplementary Figure 1, including the uncropped gels and FACS gating strategy, and Supplementary Tables 1-4.

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Daza-Martin, M., Starowicz, K., Jamshad, M. et al. Isomerization of BRCA1–BARD1 promotes replication fork protection. Nature 571, 521–527 (2019). https://doi.org/10.1038/s41586-019-1363-4

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