Isomerization of BRCA1–BARD1 promotes replication fork protection

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.

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.

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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.

Author information

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.

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

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The authors declare no competing interests.

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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.

ae, 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. Source Data

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. Source Data

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

ad, 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. Source Data

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. Source Data

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. Source Data

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.

Supplementary information

Supplementary Information

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

Reporting Summary

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