Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine 15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein 1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine 20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1–BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1–BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.
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We thank all members of the laboratory of J.R.C. for discussions; D. Durocher for plasmid reagents; N. Johnson for discussions regarding unpublished data; and T. Humphrey for comments on the manuscript. This work was funded by a Cancer Research UK (CRUK) Career Development Fellowship (C52690/A19270 to J.R.C.), which previously provided salary support to J.R.B., and CRUK Oxford Centre funding (C5255/A18085 to J.R.B. and J.R.C.). J.R.B.’s salary is provided by a Ruth L. Kirschstein NRSA Individual Postdoctoral Fellowship (F32) (NIH/NCI - F32CA239339). The laboratory of J.R.C. is also supported by the Medical Research Council (MRC - MR/R017549/1), the MRC Molecular Haematology Unit (MRC MHU, UK) and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). J.R.C. holds a Lister Institute Research Prize Fellowship. The Wellcome Centre for Human Genetics (WHG) is supported by Wellcome core award 090532/Z/09/Z. C.B. was sponsored by an ERASMUS+ internship. M.D.W.’s work is supported by the Wellcome Trust (210493), the MRC (T029471/1) and the University of Edinburgh. The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149). A.G.’s research is supported by the Lundbeck Foundation (R198-2015-269 and R313-2019-448), the European Research Council (ERC CoG no. 724436) and Independent Research Fund Denmark (7016-00042B and 4092-00404B). Research at CPR is supported by the Novo Nordisk Foundation (NNF14CC0001). We also acknowledge the BHF Centre of Research Excellence, Oxford (RE/13/1/30181), and the WHG Cellular Imaging core for equipment access and support.
A.G. is co-founder and CSO in Ankrin Therapeutics. No other authors have competing interests.
Peer review information Nature thanks Daniel Durocher, Joanna Morris 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 Fig. 1 BARD1 BRCT-mutated transgenes are stably expressed in BARD1AID/AID HCT-116 cells.
Related to Fig. 1. a, Immunoblots of whole-cell lysates collected at the indicated time points after IAA addition. Expression of the auxin-degron-targeting SCF-complex E3 ligase Oryza sativa TIR1 was induced using doxycycline (2 μg ml−1), 24 h before the depletion of endogenous BARD1–AID protein with IAA (1 mM). Representative of two biological repeats. b, Immunoblot from whole-cell lystes of BARD1 BRCT mutants screened for olaparib sensitivity. c, Immunoblot of whole cell lysates from BARD1AID/AID cells expressing the indicated transgenes. Cells were seeded in the presence of doxycycline (2 μg ml−1) and IAA (1 mM) was added after 24 h. Lysates were collected at the indicated time points after IAA addition. Representative of two biological repeats.
Extended Data Fig. 2 ARD and BRCTs in BARD1 cooperate in recruiting BRCA1 to post-replicative chromatin during homologous recombination.
Related to Fig. 2. a–c, Survival of the indicated BARD1AID/AID cell lines grown for 7 days without IAA in the presence of the indicated doses of olaparib or cisplatin. Cell lines were seeded in doxycycline (2 μg ml−1) for 24 h before olaparib or cisplatin addition. Resazurin cell viability assay, n = 3 biological experiments, mean ± s.d. d, High-content immunofluorescent microscopy of BRCA1 IRIF in H4K20me0+ BARD1AID/AID cells expressing the indicated transgenes. Cultures were grown in the presence of doxycycline (2 μg ml−1) for 24 h before IAA (1 mM) addition, irradiated 2 h later, and fixed following irradiation. Scale bar, 5 μm. Representative of n = 2 biological experiments. e, Top, quantification of BRCA1 IRIF from d. Boxes indicate the 25th–75th percentiles with the median denoted, and whiskers indicate the 10th–90th percentiles. BRCA1 foci measurements are made for nuclei in the bottom quartile of H4K20me0 integrated staining intensity (≥172 nuclei per condition). Foci quantification and H4K20me0 integrated intensity measurements were performed with CellProfiler. Bottom, mean number of BRCA1 foci per cell from two independent experiments ± s.d. f, g, Same as in d, e, respectively, in RAP80−/− cells. ≥ 179 nuclei per condition. h, i, Survival of the indicated BARD1AID/AID cell lines grown for 7 days without IAA in the presence of olaparib. Cell lines were seeded in doxycycline (2 μg ml−1) for 24 h before olaparib addition. Resazurin cell viability assay, n = 3 biological experiments, mean ± s.d
Extended Data Fig. 3 Purification of BARD1 and 53BP1 fragments and assembly of modified nucleosomes.
Related to Fig. 3. a, Western blot of HA-tagged 53BP1–BARD1 fusion proteins used in b stably expressed in BARD1AID/AID 53BP1−/− cells. b, Top, model depicting the 53BP1–BARD1 fusion protein. The fusion is a chimera composed of the 53BP1 minimal focus-forming region (amino acids 1220–1711) and BARD1 BRCTs (amino acids 555–777). Expressed form includes an N-terminal 2×HA–Flag epitope tag. Bottom, confocal immunofluorescent microscopy of 53BP1–BARD1 chimeric fusion proteins in irradiated BARD1AID/AID53BP1−/− cells. Cultures were grown in the presence of doxycycline (2 μg ml−1) for 24 h before IAA (1 mM) addition and irradiated (5 Gy) 2 h later. Cells were fixed 2 h following irradiation. Scale bar, 10 μm. Representative of n = 2 biological experiments. c, SDS–PAGE gel, stained with InstantBlue protein stain of proteins used in Fig. 3a. d, SDS–PAGE gel, stained with InstantBlue protein stain of nucleosomes used in this study. e, Native gel electrophoresis of Widom 601 DNA in isolation and wrapped with nucleosomes used in this study. f, Representative gel images from EMSA experiments quantified in Fig. 3b. H2AKc15ub-modified, or H2AKc15ub- and H4Kc20me2-modified, nucleosomes (or control DNA) were incubated with increasing concentrations of 6×His–MBP–BARD1(ARD–BRCT) or GST–53BP1(TTD–UDR). Complexes were resolved by native PAGE and visualized using Diamond DNA stain. g, SDS–PAGE gel, stained with InstantBlue protein stain of BARD1 variants used in Fig. 3c. Neighbouring lanes were loaded with two different concentrations.
Extended Data Fig. 4 BRCA1 recruitment to IRIF is RNF168-dependent, but independent of PRC1 and BRCA1–BARD1 ubiquitin ligase activity.
Related to Fig. 4. a, Model indicating the three major known sites of ubiquitin attachment on histone H2A and the genetic manipulations used to block each individually in our experiments. PRC1−/− indicates RING1A−/−RING1B−/− double knockout. b, Immunoblot of whole-cell lysates from BARD1AID/AID parental cells and RNF168−/− derivatives. Cells were seeded in the presence of doxycycline (2 μg ml−1) and IAA (1 mM) was added after 24 h. Lysates were collected 8 h after IAA addition. Representative of two biological repeats. c, Immunofluorescent microscopy of BARD1AID/AID parental cells and RNF168−/− derivatives. Cultures were seeded 24 h before irradiation (5 Gy), and fixed 2 h later. Representative of n = 3 biological experiments. Scale bar, 10 μm. d, Quantification of BRCA1 and 53BP1 IRIF from c. Boxes indicate the 25th–75th percentiles with the median denoted, and whiskers indicate the 10th–90th percentiles. BRCA1 foci measurements are made for EdU-positive nuclei. Foci quantification was performed using using CellProfiler. Significance was determined by two-sided Kruskal–Wallis H test with Dunn’s correction for multiple comparisons. ****P ≤ 0.0001. Representative of n = 3 biological replicates. e, Immunoblot of whole-cell lysates from BARD1AID/AID parental cells and RING1A−/−, RING1B−/− and RING1A−/−RING1B−/− (denoted as PRC1−/−) derivatives. Cultures were seeded 24 h before irradiation (10 Gy), and collected 2 h later. f, g, The indicated cell lines were treated as in d. Representative of n = 3 biological replicates
Extended Data Fig. 5 The β2′–β3′ loop and ARD counteract toxic 53BP1-dependent non-homologous end joining.
Related to Fig. 4. a, Immunofluorescent microscopy of RAD51 IRIF in BARD1AID/AID cells expressing the indicated transgenes. Cultures were grown in the presence of doxycycline (2 μg ml−1) for 24 h before IAA (1 mM) addition, irradiated 2 h later (5 Gy), and fixed with PFA 2 h following irradiation. Data were collected from the same experiment as Fig. 2i, j. Scale bar, 5 μm. Representative of n = 3 biological experiments. b, Top, quantification of RAD51 foci per cell from a. Per condition, ≥255 nuclei. Boxes indicate the 25th–75th percentiles with the median denoted, and whiskers indicate the 10th–90th percentiles. Significance was determined by two-sided Kruskal–Wallis H test with Dunn’s correction for multiple comparisons. ****P ≤ 0.0001, ***P = 0.0003. Data are from same experiment presented in Fig. 2i, j. BARD1AID/AID cells expressing GST and BARD1 are displayed in both for comparison. Representative of n = 3 biological experiments. Bottom, mean number of RAD51 IRIF from three independent biological experiments ± s.d. c, d, Immunoblot of whole-cell lysates from BARD1AID/AID (top) or BARD1AID/AID53BP1−/− (bottom) cells expressing the indicated transgenes. Cells were seeded in the presence of doxycycline (2 μg ml−1) and IAA (1 mM) was added after 24 h. Lysates were collected at the indicated time points after IAA addition. Representative of two biological repeats. e–h, Survival of the indicated BARD1AID/AID cell lines grown without IAA for 7 days in the presence of the indicated doses of olaparib or cisplatin. Cultures were seeded in doxycycline (2 μg ml−1) and olaparib or cisplatin was added 24 h later. Survival was measured after 7 days by resazurin cell viability assay (n = 3 biological experiments) mean ± s.d
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Becker, J.R., Clifford, G., Bonnet, C. et al. BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination. Nature 596, 433–437 (2021). https://doi.org/10.1038/s41586-021-03776-w