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BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1

Nature Structural & Molecular Biology volume 28, pages 268–277 (2021)Cite this article

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Abstract

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

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Fig. 1: RING E3 ligases ubiquitylate H2A at distinct sites on the nucleosome.
Fig. 2: Cryo-EM structure of the BRCA1-UbcH5c/BARD1/nucleosome complex.
Fig. 3: BRCA1/BARD1 RING-histone interactions.
Fig. 4: Comparison to the Ring1b-UbcH5c/Bmi1/nucleosome complex.
Fig. 5: Lysine position along the flexible C-terminal tail of H2A dictates ubiquitylation efficiency.
Fig. 6: H2A C-terminal tail dynamics in E3-E2/nucleosome complexes.
Fig. 7: Enhanced nucleosome ubiquitylation activity and binding of full-length BRCA1/BARD1.

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Data availability

The cryo-EM map of the BRCA1-UbcH5c/BARD1/nucleosome complex has been deposited to the Electron Microscopy Data Bank under accession code EMD 22581 and the atomic model to the Protein Data Bank under accession code 7JZV. NMR chemical shift assignments were deposited to the Biological Magnetic Resonance Data bank under accession code 50604. Plasmid reagents generated in this study can be obtained upon request from the corresponding author. Source data are provided with this paper.

References

  1. Tarsounas, M. & Sung, P. The antitumorigenic roles of BRCA1–BARD1 in DNA repair and replication. Nat. Rev. Mol. Cell Biol. 21, 284–299 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Densham, R. M. & Morris, J. R. Moving mountains—the BRCA1 promotion of DNA resection. Front. Mol. Biosci. 6, 79 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Hashizume, R. et al. The RING heterodimer BRCA1–BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation. J. Biol. Chem. 276, 14537–14540 (2001).

    Article  PubMed  CAS  Google Scholar 

  4. Brzovic, P. S., Rajagopal, P., Hoyt, D. W., King, M. C. & Klevit, R. E. Structure of a BRCA1–BARD1 heterodimeric RING–RING complex. Nat. Struct. Biol. 8, 833–837 (2001).

    Article  PubMed  CAS  Google Scholar 

  5. Wu, W. et al. BRCA1 ubiquitinates RPB8 in response to DNA damage. Cancer Res. 67, 951–958 (2007).

    Article  PubMed  CAS  Google Scholar 

  6. Sato, K. et al. A DNA-damage selective role for BRCA1 E3 ligase in claspin ubiquitylation, CHK1 activation and DNA repair. Curr. Biol. 22, 1659–1666 (2012).

    Article  PubMed  CAS  Google Scholar 

  7. Vázquez-Arreguín, K. et al. BRCA1 through its E3 ligase activity regulates the transcription factor Oct1 and carbohydrate metabolism. Mol. Cancer Res. 16, 439–452 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Reid, L. J. et al. E3 ligase activity of BRCA1 is not essential for mammalian cell viability or homology-directed repair of double-strand DNA breaks. Proc. Natl Acad. Sci. USA 105, 20876–20881 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Shakya, R. et al. BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity. Science 334, 525–528 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Drost, R. et al. BRCA1 ring function is essential for tumor suppression but dispensable for therapy resistance. Cancer Cell 20, 797–809 (2011).

    Article  PubMed  CAS  Google Scholar 

  11. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Densham, R. M. & Morris, J. R. The BRCA1 ubiquitin ligase function sets a new trend for remodelling in DNA repair. Nucleus 8, 116–125 (2017).

    Article  PubMed  CAS  Google Scholar 

  13. Thakar, A., Parvin, J. D. & Zlatanova, J. BRCA1/BARD1 E3 ubiquitin ligase can modify histones H2A and H2B in the nucleosome particle. J. Biomol. Struct. Dyn. 27, 399–405 (2010).

    Article  PubMed  CAS  Google Scholar 

  14. Kalb, R., Mallery, D. L., Larkin, C., Huang, J. T. J. & Hiom, K. BRCA1 is a histone-H2A-specific ubiquitin ligase. Cell Rep. 8, 999–1005 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Zhu, Q. et al. BRCA1 tumour suppression occurs via heterochromatin-mediated silencing. Nature 477, 179–184 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Kim, B.-J. et al. The histone variant MacroH2A1 is a BRCA1 ubiquitin ligase substrate. Cell Rep. 19, 1758–1766 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Stewart, M. D. et al. BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes. Proc. Natl Acad. Sci. USA 115, 1316–1321 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Uckelmann, M. et al. USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A. Nat. Commun. 9, 229 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Mattiroli, F. et al. RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling. Cell 150, 1182–1195 (2012).

    Article  PubMed  CAS  Google Scholar 

  20. Mattiroli, F., Uckelmann, M., Sahtoe, D. D., van Dijk, W. J. & Sixma, T. K. The nucleosome acidic patch plays a critical role in RNF168-dependent ubiquitination of histone H2A. Nat. Commun. 5, 3291 (2014).

    Article  PubMed  CAS  Google Scholar 

  21. Fradet-Turcotte, A. et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Wilson, M. D. et al. The structural basis of modified nucleosome recognition by 53BP1. Nature 536, 100–103 (2016).

    Article  PubMed  CAS  Google Scholar 

  23. Wang, H. et al. Role of histone H2A ubiquitination in polycomb silencing. Nature 431, 873–878 (2004).

    Article  PubMed  CAS  Google Scholar 

  24. Cao, R., Tsukada, Y. & Zhang, Y. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing. Mol. Cell 20, 845–854 (2005).

    Article  PubMed  CAS  Google Scholar 

  25. Tamburri, S. et al. Histone H2AK119 mono-ubiquitination is essential for polycomb-mediated transcriptional repression. Mol. Cell 77, 840–856 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Vissers, J. H. A., van Lohuizen, M. & Citterio, E. The emerging role of Polycomb repressors in the response to DNA damage. J. Cell Sci. 125, 3939–3948 (2012).

    Article  PubMed  CAS  Google Scholar 

  27. McGinty, R. K., Henrici, R. C. & Tan, S. Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514, 591–596 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Horn, V. et al. Structural basis of specific H2A K13/K15 ubiquitination by RNF168. Nat. Commun. 10, 1751 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Brzovic, P. S. et al. Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex. Proc. Natl Acad. Sci. USA 100, 5646–5651 (2003).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Anderson, C. J. et al. Structural basis for recognition of ubiquitylated nucleosome by Dot1L methyltransferase. Cell Rep. 26, 1681–1690 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Valencia-Sánchez, M. I. et al. Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. Mol. Cell 74, 1010–1019 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Song, Y. et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013).

    Article  PubMed  CAS  Google Scholar 

  33. Yang, J. et al. Improved protein structure prediction using predicted interresidue orientations. Proc. Natl Acad. Sci. USA 117, 1496–1503 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. McGinty, R. K. & Tan, S. Recognition of the nucleosome by chromatin factors and enzymes. Curr. Opin. Struct. Biol. 37, 54–61 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Taherbhoy, A. M., Huang, O. W. & Cochran, A. G. BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase. Nat. Commun. 6, 7621 (2015).

    Article  PubMed  Google Scholar 

  36. Wood, K., Tellier, M. & Murphy, S. DOT1L and H3K79 methylation in transcription and genomic stability. Biomolecules 8, 11 (2018).

    Article  PubMed Central  CAS  Google Scholar 

  37. Nguyen, A. T. & Zhang, Y. The diverse functions of Dot1 and H3K79 methylation. Genes Dev. 25, 1345–1358 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Huyen, Y. et al. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature 432, 406–411 (2004).

    Article  PubMed  CAS  Google Scholar 

  39. Wysocki, R. et al. Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9. Mol. Cell. Biol. 25, 8430–8443 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Tate, J. G. et al. COSMIC: the catalogue of somatic mutations in cancer. Nucleic Acids Res. 47, D941–D947 (2019).

    Article  PubMed  CAS  Google Scholar 

  41. Shah, S., Verma, T., Rashid, M., Gadewal, N. & Gupta, S. Histone H2A isoforms: potential implications in epigenome plasticity and diseases in eukaryotes. J. Biosci. 45, 4 (2020).

    Article  PubMed  CAS  Google Scholar 

  42. Nakamura, K. et al. H4K20me0 recognition by BRCA1–BARD1 directs homologous recombination to sister chromatids. Nat. Cell Biol. 21, 311–318 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Becker, J. R. et al. BARD1 links histone H2A Lysine-15 ubiquitination to initiation of BRCA1-dependent homologous recombination. Preprint at bioRxiv https://doi.org/10.1101/2020.06.01.127951 (2020).

  44. Masuda, T., Xu, X., Dimitriadis, E. K., Lahusen, T. & Deng, C.-X. ‘DNA binding region’ of BRCA1 affects genetic stability through modulating the intra-S-phase checkpoint. Int. J. Biol. Sci. 12, 133–143 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Streich, F. C. Jr & Lima, C. D. Capturing a substrate in an activated RING E3/E2–SUMO complex. Nature 536, 304–308 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Scott, D. C. et al. Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. Cell 157, 1671–1684 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Brown, N. G. et al. RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex. Proc. Natl Acad. Sci. USA 112, 5272–5279 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M. & Wolberger, C. Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat. Struct. Mol. Biol. 13, 915–920 (2006).

    Article  PubMed  CAS  Google Scholar 

  50. Pruneda, J. N. et al. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol. Cell 47, 933–942 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Paull, T. T. et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895 (2000).

    Article  PubMed  CAS  Google Scholar 

  52. Cooper, S. et al. Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2. Nat. Commun. 7, 13661 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Savage, K. I. & Harkin, D. P. BRCA1, a ‘complex’ protein involved in the maintenance of genomic stability. FEBS J. 282, 630–646 (2015).

    Article  PubMed  CAS  Google Scholar 

  54. Belotserkovskaya, R. et al. PALB2 chromatin recruitment restores homologous recombination in BRCA1-deficient cells depleted of 53BP1. Nat. Commun. 11, 819 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Carrion-Vazquez, M., Marszalek, P. E., Oberhauser, A. F. & Fernandez, J. M. Atomic force microscopy captures length phenotypes in single proteins. Proc. Natl Acad. Sci. USA 96, 11288–11292 (1999).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Aiyar, A., Xiang, Y. & Leis, J. Site-directed mutagenesis using overlap extension PCR. Methods Mol. Biol. 57, 177–191 (1996).

    PubMed  CAS  Google Scholar 

  57. Christensen, D. E., Brzovic, P. S. & Klevit, R. E. E2–BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat. Struct. Mol. Biol. 14, 941–948 (2007).

    Article  PubMed  CAS  Google Scholar 

  58. Lazar, G. A., Desjarlais, J. R. & Handel, T. M. De novo design of the hydrophobic core of ubiquitin. Protein Sci. 6, 1167–1178 (1997).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Lee, Y.-T., Gibbons, G., Lee, S. Y., Nikolovska-Coleska, Z. & Dou, Y. One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer. Protein Expr. Purif. 110, 89–94 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Luger, K., Rechsteiner, T. J. & Richmond, T. J. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999).

    Article  PubMed  CAS  Google Scholar 

  61. Dhall, A. et al. Sumoylated human histone H4 prevents chromatin compaction by inhibiting long-range internucleosomal interactions. J. Biol. Chem. 289, 33827–33837 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Simon, M. D. et al. The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128, 1003–1012 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Dyer, P. N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23–44 (2004).

    Article  PubMed  CAS  Google Scholar 

  64. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  PubMed  CAS  Google Scholar 

  65. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  PubMed  CAS  Google Scholar 

  66. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  PubMed  CAS  Google Scholar 

  67. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  PubMed  CAS  Google Scholar 

  68. Johnson, B. A. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol. Biol. 278, 313–352 (2004).

    PubMed  CAS  Google Scholar 

  69. Morrison, E. A., Bowerman, S., Sylvers, K. L., Wereszczynski, J. & Musselman, C. A. The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome. Elife 7, e31481 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Tan (Penn State University) for sharing Ring1b/Bmi1plasmids and L. Kay (University of Toronto) for sharing the 153-base-pair Widom 601 repeat plasmid. We thank N. Zheng for his insightful feedback on the manuscript and sharing laboratory equipment; T. Hinds for assistance with ITC experiments; P. Hsu for advice on nucleosome complexes; J. Quispe and Q. Beedle for technical assistance with cryo-EM data collection; and L. Walls, A. Borst and D. Veesler for advice on single-particle cryo-EM data processing. S.R.W. acknowledges support from the NIH (T32GM008268). A.L.B. was supported by the NIH (T32GM008268 and 1F31EY030732). D.P.F. was supported through the Human Frontiers Science Program (RGP0061/2019). J.M.H. was supported by the NIH (T32GM007270). W.Z. was supported by a V Scholar Grant (V2019.Q13) from the V Foundation and a Young Investigator Award from the Max and Minnie Tomerlin Voelcker Fund. F.D. was supported by the NIH (GM123089). R.E.K. is the Edmond H. Fischer/Washington Research Foundation Endowed Chair in Biochemistry and is supported by the NIH (GM088055).

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  1. Mikaela D. Stewart

    Present address: Department of Biology, Texas Christian University, Fort Worth, TX, USA

Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, WA, USA

    Samuel R. Witus, Anika L. Burrell, Daniel P. Farrell, Jianming Kang, Jesse M. Hansen, Alex Pravat, Lisa M. Tuttle, Mikaela D. Stewart, Peter S. Brzovic, Frank DiMaio, Justin M. Kollman & Rachel E. Klevit

  2. Institute for Protein Design, University of Washington, Seattle, WA, USA

    Daniel P. Farrell & Frank DiMaio

  3. Department of Chemistry, University of Washington, Seattle, WA, USA

    Jianming Kang & Champak Chatterjee

  4. Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX, USA

    Meiling Wang & Weixing Zhao

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Contributions

S.R.W., M.D.S., P.S.B. and R.E.K. conceived the project, designed the experiments and analyzed data. S.R.W. prepared the cryo-EM sample, imaged and analyzed data with A.L.B. and J.M.H. under the supervision of J.M.K. D.P.F. built the atomic model under the supervision of F.D. with input from S.R.W. J.K. generated methylated nucleosomes under the supervision of C.C. S.R.W. and L.M.T. conducted and analyzed the NMR experiments. M.W. and W.Z. produced full-length BRCA1/BARD1 and performed binding assays. S.R.W. and A.P. purified proteins, performed assays and analyzed data. S.R.W., P.S.B. and R.E.K. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Rachel E. Klevit.

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The authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Specificity of BRCA1/BARD1-dependent nucleosome ubiquitylation and validation of the E3-E2 chimera.

a, Western blot analysis of all four histone subunits from the same nucleosome ubiquitylation reaction. b, Native-gel EMSA measuring NCP binding of BRCA1-UbcH5c/BARD1 constructs with various Gly/Ser-repeat linker lengths. The E3-E2 chimera with a seven-residue linker was used for structure determination and all subsequent experiments. c, Nucleosome ubiquitylation assays using BRCA1/BARD1 with indicated E2 enzymes. d, Coomassie-stained gel under reducing or non-reducing conditions of an E2 charging reaction using the BRCA1-UbcH5c/BARD1 chimera. e, Nucleosome ubiquitylation activity of BRCA1/BARD1 with UbcH5c in trans or the BRCA1-UbcH5c/BARD1 chimera with wild-type (WT) or Lys125/127/129Arg (3KR) NCP substrates. Data in panels c and e are representative of n=2 independent experiments. Uncropped gels/blots in panels a-e are available as source data.

Source data

Extended Data Fig. 2 Purification of a stable BRCA1-UbcH5c/BARD1/nucleosome complex.

a, Coomassie-stained gel of an E2 charging reaction using UbcH5cC85K and BRCA1-UbcH5cC85K/BARD1. b, Size exclusion chromatography (SEC) of NCPs (black) with excess BRCA1-UbcH5c/BARD1 (purple) or BRCA1-UbcH5cC85K/BARD1 (green) with 35 mM NaCl in SEC buffer. c, Coomassie-stained gels of fractions from SEC binding experiments shown in panel b. d, SEC of NCPs with excess BRCA1-UbcH5cC85K/BARD1 with 100 mM NaCl (black) or 25 mM NaCl (green) in SEC running buffer. e, Coomassie-stained gels of fractions from SEC binding experiments shown in panel d. f, Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of NCPs (black) and the BRCA1-UbcH5cC85K/BARD1/NCP complex (green). Dashed lines report MALS molecular weight (MW) data. The MW value reported is the average MW from light scattering ± 1-s.d. where error is a measure of statistical consistency of light scattering data and not an absolute bound on the error of MW. The expected MW is 203.7 kDa for the NCP and 287.2 kDa for the complex bound with 2:1 stoichiometry. Uncropped gels in panels a, c, and e are available as source data.

Source data

Extended Data Fig. 3 Cryo-EM processing workflow.

a, Flow chart of data processing steps. Objects with dark borders represent solvent masks and are not density maps. b, Representative motion-corrected micrograph from data set. White scale bar is 100 nm.

Extended Data Fig. 4 Cryo-EM validation and example density.

a, Local resolution estimate from CryoSparc after non-uniform refinement at the FSC=0.143 cutoff plotted on the final density modified map of the whole complex (left) and close-up on the RING-histone interface (right). b, Half-map Fourier shell correlation curves from CryoSparc non-uniform refinement (red) and after density modification (blue). c, Euler angle distribution of refined particle subset used in final cryo-EM reconstruction. d, Main chain trace of the BRCA1 RING, BARD1 RING, four-helix bundle, and UbcH5c fit into the cryo-EM map. The active site position of UbcH5c (Lys85) Cα is shown as a sphere. e, Subset of important histone regions fit into the cryo-EM map with side chains shown (except for H2A C-terminal tail).

Extended Data Fig. 5 Extensive analysis of the BRCA1/BARD1 RING-histone interface.

a, b, c, Close-up view of BRCA1 and BARD1 RING domains in the complex showing side chains of residues mutated in nucleosome ubiquitylation assays in panels d-g and j-k, d, Representative nucleosome ubiquitylation assay using the indicated BRCA1 RING mutants. e, Quantified nucleosome ubiquitylation assays for BRCA1 mutants shown in panel d. (f, g) Same experimental set-up as panels d and e using the indicated BARD1 mutants. h, ITC binding data for BRCA1/BARD1 and indicated mutants with NCPs. Summarized data are also shown in Fig. 3e. i, 1H15N-TROSY-HSQC spectra of wild-type (WT) BRCA1/BARD1 and BRCA1(WT)/BARD1(Trp91Ala). j, k, Same experimental set-up as panels d and e using BARD1 mutants reported on the COSMIC database. l, E2~Ub lysine discharge assay using the indicated BRCA1/BARD1 mutants. m, Quantified E2~Ub lysine discharge assays using the BRCA1/BARD1 mutants shown in panel l. n, o, Same experimental set-up as panels l and m using BARD1 COSMIC mutants. Quantified nucleosome ubiquitylation assay data show the mean and error bars are ± 1-s.d. of n=3 independent experiments at 10-minute (gray bars) and 30-minute (colored bars) time points. Statistical analysis is compared to wild-type at the 10-minute time point. Quantified E2 lysine discharge data show the mean and error bars are ± 1-s.d. of n=3 independent experiments at 2-, 6- and 10- minute time points (light, medium, and dark gray bars). Statistical comparisons are indicated with lines above the graphs. All p-values were calculated using a two-tailed Student’s t-test (* p≤0.05, ** p≤0.005, ns = not significant). Uncropped gels/blots in panels d, f, j, l, and n and data from graphs in panels e, g, k, m, and o are available as source data.

Source data

Extended Data Fig. 6 Comparison of nucleosome requirements for H2A-modifying E3s.

a, Comparison of the conserved BRCA1 and Ring1b arginine anchor motif interactions (PDB: 4R8P). Complexes were aligned by H2B on the bound face of the NCP. H2A from the Ring1b-UbcH5c/Bmi1 nucleosome complex is shown in gray. b, Nucleosome ubiquitylation assays using the indicated H2A/H2B acidic patch NCP mutants with BRCA1/BARD1 (top) and Ring1b/Bmi1 (bottom). c, Comparison of interactions between the RING domains of BRCA1/BARD1 and Ring1b/Bmi1 with the H2B αC helix residues assayed in panels d and e. d, Representative nucleosome ubiquitylation assays using the indicated H2B αC helix NCP mutants with BRCA1/BARD1 (top) or Ring1b/Bmi1 (bottom). e, Quantified assays using the NCP mutants and E3s from panel d. f, g, Same experimental set-up as in panels d and e using H2B/H4 cleft NCP mutants. h, BARD1-histone binding interface with side chain density for H3 Phe78 and Lys79 shown as semi-transparent surface. i, j, Same experimental set-up as panels d and e using H3 Lys79Ala NCP mutant. k, l, m, Representative nucleosome ubiquitylation assays using H3 Lys79me1 and Lys79me2 mimetic NCP substrates with the indicated E3s. n, o, p, Quantified assays using H3 Lys79 methylation mimetic nucleosome substrates and the indicated E3s. Quantified ubiquitylation assays show the mean and error bars are ± 1-s.d. of n=3 independent experiments (except panel g where n=6 for wild-type) plotted for 10-minute (gray bars) and 30-minute (colored bars) time points. P-values for each mutant were calculated using a two-tailed Student’s t-test compared to wild-type (* p≤0.05, ** p≤0.005, ns = not significant). Panels g (top), and n are also shown in Fig. 3. Uncropped blots in panels b, d, f, i, k, l, and m and data from graphs in panels e, g, j, n, o, and p are available as source data.

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Extended Data Fig. 7 Comparison to the unbound RING heterodimer and Ring1b-UbcH5c/Bmi1/nucleosome complexes.

a, Structural alignment of the BRCA1/BARD1 RING heterodimers from the NCP complex and the unbound structure (PDB: 1JM7, NMR ensemble model 1) aligned by the four-helix bundle. b, Structural alignment of the NCP from the indicated complexes by H2B on the E3-E2-bound side. c, Structural alignment of BRCA1-UbcH5c and Ring1b-UbcH5c from E3-E2/NCP complexes. d, Comparison of BRCA1-bound and Ring1b-bound UbcH5c in NCP complexes showing side chains mutated in panels g and h. Some atoms for Arg72 and Lys128 are not modelled in the Ring1b-bound structure. Green spheres are Cα of H2A Lys118. e, Analysis of NCP binding by the BRCA1/BARD1 RING heterodimer and the BRCA1-UbcH5c/BARD1 (WT UbcH5c active site) chimera by ITC. Data are representative of n=2 experiments. f, Comparison of NCP binding strength for BRCA1/BARD1 and Ring1b/Bmi1 RING heterodimer and E3-E2 chimeras. Affinities for BRCA1/BARD1 were determined using ITC while affinities for Ring1b/Bmi1 were measured using a fluorescence based binding assay27. g, Representative nucleosome ubiquitylation assays using BRCA1/BARD1 or Ring1b/Bmi1 with indicated UbcH5c mutants. h, Quantification of nucleosome ubiquitylation using the UbcH5c mutants and E3s from panel g. Data show the mean and error bars are ± 1-s.d. of n=3 independent experiments plotted for 10-minute (gray bars) and 30-minute (colored bars) time points of the reactions. P-values for each mutant were calculated using a two-tailed Student’s t-test compared to wild-type (* p≤0.05, ** p≤0.005, ns = not significant). Uncropped blots in panel g and data from graphs in panel h are available as source data.

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Extended Data Fig. 8 NMR analysis of E3-E2/nucleosome complexes.

a, 1H15N-TROSY-HSQC spectrum of 2H15N13C-H2A in NCP (~180 µM sample) with residue assignments plotted. Signals with asterisks were not able to be assigned. Residues 120# and 120## appear to be alternate confirmations of 120 based on Cα chemical shifts of residues 119 and 120 in the HNCA and HNCOCA spectra. Assignment of the minor population for 6# was based on the similar back bone H, N, and Cα chemical shifts as compared to the major residue 6 population. b, Sequence of H2A N- and C-terminal tails observable in NMR spectrum in panel a with assigned residues bolded. c, Overlay of 1H15N-TROSY-HSQC spectra of 2H15N-H2A in NCP, with BRCA1-UbcH5cC85K/BARD1 added at increasing concentrations. d, Quantification of signal broadening from spectra in panel c, as a function of BRCA1-UbcH5cC85K/BARD1 added, normalized to the first titration point (60 μM addition). Signal behavior clusters into two groups, where resonances from the extreme N- and C- terminal tails lose signal at a similar rate (cluster 1). e, Overlay of 1H15N-TROSY-HSQC spectra of 2H15N-H2A in NCP (black, bottom), with BRCA1-UbcH5cC85K/BARD1 added (red, middle), or BRCA1-UbcH5c/BARD1 (blue, top). f, Quantification of intensity for H2A signals from spectra in panel e comparing the bound complexes to the apo reference spectrum (Icom/Iref). g, Overlay of 1H15N-TROSY-HSQC spectra of 2H15N-H3 in NCP (black, bottom), with BRCA1-UbcH5cC85K/BARD1 added (red, middle), or Ring1b-UbcH5c/Bmi1 (pink, top). h, Quantification of intensity for H2A signals from spectra in panel g comparing the bound complexes to the apo reference spectrum (Icom/Iref). Concentrations of E3-E2 complexes added as well as labelled NCPs in the experiment are indicated in the figure. Data from graphs in panels d, f, and h are available as source data.

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Extended Data Fig. 9 Biochemical purity and H2A specificity of full-length BRCA1/BARD1.

a, Coomassie-stained gel of purified full-length BRCA1/BARD1. b, Nucleosome ubiquitylation assay using full-length BRCA1/BARD1 and wild-type (WT) or H2A Lys125/127/129Arg (3KR) nucleosome substrates. Uncropped gels/blots in panels a and b are available as source data.

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Extended Data Fig. 10 Compatibility of closed E2~Ub conformations.

a, Structural overlay of the BRCA1-UbcH5c/BARD1/nucleosome complex with indicated RING/E2-Ub crystal structures aligned to the RING domain of BRCA1. b, Comparison of UbcH5c location in the nucleosome complex (purple) and indicated reported RING/E2-Ub structures from panel a with cryo-EM density for UbcH5c shown as gray volume. Density for the E2 is shown from a map that was locally filtered in CryoSparc to decrease excessive noise introduced from map sharpening in this region.

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Witus, S.R., Burrell, A.L., Farrell, D.P. et al. BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1. Nat Struct Mol Biol 28, 268–277 (2021). https://doi.org/10.1038/s41594-020-00556-4

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