Abstract
The opposing activities of 53BP1 and BRCA1 influence pathway choice in DNA double-strand-break repair. How BRCA1 counteracts the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1–BARD1 required for priming ubiquitin transfer from E2∼ubiquitin and demonstrate that BRCA1–BARD1's ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitination by BRCA1–BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1-deficient cells. BRCA1–BARD1's function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin and optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning, and the need for SMARCAD1 in olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus, BRCA1–BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Long, D.T. & Walter, J.C. A novel function for BRCA1 in crosslink repair. Mol. Cell 46, 111–112 (2012).
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).
Jiang, Q. & Greenberg, R.A. Deciphering the BRCA1 tumor suppressor network. J. Biol. Chem. 290, 17724–17732 (2015).
Panier, S. & Boulton, S.J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18 (2014).
Bunting, S.F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).
Escribano-Díaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872–883 (2013).
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).
Kalb, R., Mallery, D.L., Larkin, C., Huang, J.T. & Hiom, K. BRCA1 is a histone-H2A-specific ubiquitin ligase. Cell Rep. 8, 999–1005 (2014).
Zhu, Q. et al. BRCA1 tumour suppression occurs via heterochromatin-mediated silencing. Nature 477, 179–184 (2011).
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).
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).
Drost, R. et al. BRCA1 RING function is essential for tumor suppression but dispensable for therapy resistance. Cancer Cell 20, 797–809 (2011).
Plechanovová, A. et al. Mechanism of ubiquitylation by dimeric RING ligase RNF4. Nat. Struct. Mol. Biol. 18, 1052–1059 (2011).
Dou, H., Buetow, L., Sibbet, G.J., Cameron, K. & Huang, D.T. Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3. Nat. Struct. Mol. Biol. 20, 982–986 (2013).
Metzger, M.B., Pruneda, J.N., Klevit, R.E. & Weissman, A.M. RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochim. Biophys. Acta 1843, 47–60 (2014).
Morris, J.R. et al. Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility. Hum. Mol. Genet. 15, 599–606 (2006).
Mallery, D.L., Vandenberg, C.J. & Hiom, K. Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains. EMBO J. 21, 6755–6762 (2002).
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).
Ismail, I.H., McDonald, D., Strickfaden, H., Xu, Z. & Hendzel, M.J. A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks. J. Biol. Chem. 288, 26944–26954 (2013).
Morris, J.R., Keep, N.H. & Solomon, E. Identification of residues required for the interaction of BARD1 with BRCA1. J. Biol. Chem. 277, 9382–9386 (2002).
Boersma, V. et al. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end resection. Nature 521, 537–540 (2015).
Xu, G. et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature 521, 541–544 (2015).
Wang, J. et al. PTIP associates with Artemis to dictate DNA repair pathway choice. Genes Dev. 28, 2693–2698 (2014).
Chapman, J.R., Sossick, A.J., Boulton, S.J. & Jackson, S.P. BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. J. Cell Sci. 125, 3529–3534 (2012).
Kakarougkas, A. et al. Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection. Nucleic Acids Res. 41, 10298–10311 (2013).
Cejka, P. DNA end resection: nucleases team up with the right partners to initiate homologous recombination. J. Biol. Chem. 290, 22931–22938 (2015).
Cruz-García, A., López-Saavedra, A. & Huertas, P. BRCA1 accelerates CtIP-mediated DNA-end resection. Cell Rep. 9, 451–459 (2014).
Shibata, A. et al. Role of ATM and the damage response mediator proteins 53BP1 and MDC1 in the maintenance of G(2)/M checkpoint arrest. Mol. Cell. Biol. 30, 3371–3383 (2010).
Tomimatsu, N. et al. Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions. DNA Repair (Amst.) 11, 441–448 (2012).
Grabarz, A. et al. A role for BLM in double-strand break repair pathway choice: prevention of CtIP/Mre11-mediated alternative nonhomologous end-joining. Cell Rep. 5, 21–28 (2013).
Chen, X. et al. The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends. Nature 489, 576–580 (2012).
Costelloe, T. et al. The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature 489, 581–584 (2012).
Eapen, V.V., Sugawara, N., Tsabar, M., Wu, W.H. & Haber, J.E. The Saccharomyces cerevisiae chromatin remodeler Fun30 regulates DNA end resection and checkpoint deactivation. Mol. Cell. Biol. 32, 4727–4740 (2012).
Neves-Costa, A., Will, W.R., Vetter, A.T., Miller, J.R. & Varga-Weisz, P. The SNF2-family member Fun30 promotes gene silencing in heterochromatic loci. PLoS One 4, e8111 (2009).
Shih, S.C. et al. A ubiquitin-binding motif required for intramolecular monoubiquitylation, the CUE domain. EMBO J. 22, 1273–1281 (2003).
Hickson, I. et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159 (2004).
Byeon, B. et al. The ATP-dependent chromatin remodeling enzyme Fun30 represses transcription by sliding promoter-proximal nucleosomes. J. Biol. Chem. 288, 23182–23193 (2013).
Awad, S., Ryan, D., Prochasson, P., Owen-Hughes, T. & Hassan, A.H. The Snf2 homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme. J. Biol. Chem. 285, 9477–9484 (2010).
Rowbotham, S.P. et al. Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1. Mol. Cell 42, 285–296 (2011).
Buchwald, G. et al. Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b. EMBO J. 25, 2465–2474 (2006).
Huang, A. et al. Symmetry and asymmetry of the RING-RING dimer of Rad18. J. Mol. Biol. 410, 424–435 (2011).
Kappo, M.A. et al. Solution structure of RING finger-like domain of retinoblastoma-binding protein-6 (RBBP6) suggests it functions as a U-box. J. Biol. Chem. 287, 7146–7158 (2012).
Boutell, C., Sadis, S. & Everett, R.D. Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro. J. Virol. 76, 841–850 (2002).
Lukas, C., Falck, J., Bartkova, J., Bartek, J. & Lukas, J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 5, 255–260 (2003).
Acknowledgements
Grant funding for this project was as follows. CRUK: C8820/A19062 (R.M.D., A.G., H.R.S. and J.B.), C302/A14532 and C1206/A11978 (F.Z.W. and L.H.P.); Breast Cancer Campaign: 2010MayPR01 (J.S.) and 2013NovPR132 (S.B.-R.); Engineering and Physical Sciences Research Council EP/L016346/1 (R.N.); University of Birmingham (A.F., B.J. and R.M.D.); University of Sussex (R.A.B.); HEFCE (J.R.M., R.N. and N.H.K.). We thank J. Stark (City of Hope) for U20S-DR3 and U20S-EJ5 cells and the I-SceI plasmid; R. Everett (MRC Virology Unit, Glasgow) for UBE2D1 cDNA and the myc-His-ubiquitin construct; T. Sixma and M. van Lohuizen, (both NKI Netherlands) for the Ring1b159/Bmi1109 coexpression construct and BMI1-GFP; A. Zlatanou (University of Birmingham) for HA-Rad18; G. Stewart (University of Birmingham) for Flp-In HeLa cells and anti-MDC1 antibody; R. Katz (Fox Chase Cancer Center) for cryptic EGFP HeLa cells; and J. Tainer (Scripps Research Institute) for PFM01 inhibitor. Determination of the structure of the TRIM37 domain was supported by an NIH Protein Structure Initiative grant to G.T. Montelione, J.F. Hunt and the Northeast Structural Genomics Consortium. We thank R. Hay for useful discussions and T. Sixma and M. Uckelmann for insights into the contribution of H2A-Ub interactions in purified nucleosomes.
Author information
Authors and Affiliations
Contributions
R.M.D. performed structural analysis, generated proteins, performed cell and biochemical experiments, designed experiments and interpreted data. A.J.G. generated constructs and cell lines, and performed colony survival experiments. H.R.S. performed resection experiments and fluorescence microscopy. J.S. generated BARD1 cell lines and performed immunoprecipitation experiments. R.A.B. performed high-resolution BRCA1 and 53BP1 microscopy and analysis. A.F. generated RING1B–BMI1 proteins and performed biochemistry. B.J. quantified foci. M.D.-M. generated BRCA1 cell lines. S.B.-R. and J.B. provided technical support and yeast experiments. L.H.P., R.N., N.H.K. and F.Z.W. provided supervisory help and data interpretation. J.R.M. and R.M.D. wrote the paper. A.J.G., H.R.S. and M.D.-M. commented on the paper and ongoing research. J.R.M. contributed to data interpretation and directed the project.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 A basic residue of BARD1 promotes Ub transfer from BRCA1-E2~Ub.
A Scan of external facing BARD1 residues 91-99 for impact on Ub chain formation catalysed by the BRCA1-BARD1 ligase. His-tagged BRCA1 (amino acids 1–300) and either His-tagged WT-BARD1 (amino acids 26–142) or BARD1 bearing the amino acid substitutions shown were co-purified from bacteria, and checked for parity before being subjected to a Ub ligase assay with free Ub and UBE2D1 enzyme.
B Auto-ubiquitination of BRCA1 is impaired by substitution of R99-BARD1. BRCA1 and WT or mutant BARD1 were co-purified from bacteria, before incubation with UBE2D1 and K-less-Ub. The western shows BRCA1-Ub by blotting for Ub (top) and BRCA1 (bottom).
C R99K- substitution of BARD1 has little impact on heterodimer ligase activity. BRCA1 and WT or mutant BARD1 were co-purified from bacteria, and checked for parity before being subjected to Ub ligase assay with the UBE2D1 enzyme and Ub.
D R99E-BARD1 and R99A-BARD1 heterodimers are poorly active with all UBE2D family members. BRCA1 and either WT or mutant BARD1 were co-purified from bacteria, checked for parity before being subjected to a Ub ligase assay with the UBE2D family: D1(UbcH5a), D2 (UbcH5b) and D3 (UbcH5c) enzymes.
E R99E-BARD1 expressed and purified at a 1:1 ratio with BRCA1. Top panel illustrates the bi-cistronic vector used (green triangles represent 6-histidine tag). Proteins were purified using a nickel column and run on a gel before being Coomassie stained.
F Contribution of BARD1-R99 to the E2~Ub interaction with BRCA1. VP16-BRCA11-300 and full length BARD1, were transformed with WT or mutant E2 enzyme LexA-UBE2D1. Yeast also express endogenous Ub. Reduced growth is seen with the E2 catalytic cysteine mutant, C85A, which prevents thioester bonding with Ub, indicating that the heterodimer interacts more strongly with a Ub loaded E2. A slight increase in growth is seen with the C85K mutant, which is reported to increase Ub~E2 stability by preventing Ub transfer (11). I26A-BRCA1 (middle panel) severely impairs E2 interactions (1), and likewise R99E-BARD1 reduces E2 interactions. ‘Trans-c’ is growth on media lacking leucine, tryptophan and adenine, selecting for all three plasmids. Growth on media also lacking histidine in increasing concentrations of the HIS3 competitive inhibitor, 3AT, is indicative of HIS3 transcription driven by VP16-BRCA1:UBE2D1-LexA interaction.
G Modelling of BRCA1-BARD1 on RNF4-RNF4-Ub~E2. The RING domain of BRCA1 (PDB: 1JM7 chain A) was superimposed on the RING domain of RNF4-RNF4 contacting the E2, UBE2D2 (S22R and C85K)–Ub complex (PDB: 4AP4), before removal of the RNF4 structures. BRCA1 is shown in green, BARD1 in orange and UBE2D2 in blue and Ub in brown. The image below is a 90 degree rotation about the horizontal. R99-BARD1 side chain is shown in pink and the Ub D32 side-chain in yellow. Zinc ions are filled spheres (black).
H Ability of WT and R99E mutant heterodimer to discharge Ub from a loaded E2 (UBE2D3). E2 enzyme was first charged with Ub in the absence of an E3 and substrate, and then incubated with excess lysine and the heterodimer for the times shown before stopping the reaction. E2~Ub dimer was quantified, and the mean across three experiments shown, bars = S.E.
Supplementary Figure 2 Other type 1 RING E3 ligases require arginine or lysine residues on the partner protomer.
A Structural sequence alignment of the RING regions highlighting K/R residues (green outline box) and cysteine /histidine residues of the RING (black outline box), α-helices are in red, β-sheets in blue.
B Purification strategy of RING1B-BMI1, and demonstration of equivalent purification of mutant and WT forms of heterodimer.
C Inhibition of Ub-signalling in the DNA damage response by ectopic expression of K73E-BMI1 but not WT-BMI1. Cells were transfected with GFP vector, WT-BMI1-GFP or K73E-BMI1-GFP, irradiated with 5 Gy and fixed an hour later before staining with antibodies to γH2AX, pATM, MDC1, 53BP1, and Ubiquitin conjugates (FK2-Ub). Representative images are shown above for GFP vector (top), WT-BMI1 (Middle) or K73E-BMI1 (bottom) for staining for MDC1 (right), where no impact of K73E-BMI1 is seen, FK2-Ub and 53BP1, where expression of K73E-BMI1 is inhibitory to protein recruitment to foci. White arrows indicate GFP positive cells. Data is quantified in C.
D The % BMI1-GFP positive HeLa cells with >10 foci of each type was quantified relative to % GFP only control (graph below, 100 cells per set, 3 independent experiments, bars = S.E).
E Complementation of BMI1 siRNA treated cells with siRNA resistant WT BMI1 or with K73E-BMI1 in gene conversion of a substrate integrated into U20S cells after I-Sce-1 transfection. NTC is non-targeting control siRNA. (DR3-GFP substrate and repair products are illustrated above). (Each assay 3 technical repeats, 3 experiments, bars = S.E. * indicates p<0.05).
Supplementary Figure 3 BARD1 L44R and R99E separation-of-function variants.
A Transfection with BARD1 siRNA and complementation with WT-BARD1 or R99E-BARD1 supports the formation of endogenous BRCA1 foci after irradiation (IR). In contrast BRCA1 foci do not form in cells complemented with L44R-BARD1. BARD1 complemented cells were irradiated (5 Gy) and allowed an hour to recover before fixation and staining for BRCA1. Quantification of BRCA1 foci at times shown after IR treatment in cells complemented with BARD1 proteins show in the graph, right (bars = S.E., n=100 cells).
B WT-BARD1 and R99E-BARD1 stabilize and co-purify endogenous BRCA1, whereas L44R-BARD1 does not. Immunoprecipitation of Flag-bound proteins from 293 cells expressing WT, R99E or L44R substituted RFP-Flag-BARD1, input levels, from Whole Cell Extract (WCE) is shown. (Note the increased endogenous BRCA1 on WT or R99E BARD1 expression).
C WT-BARD1 and R99E-BARD1 heterodimers are stable. BARD1 variants were expressed in 293 cells and treated with Cycloheximide (CHX) for the times shown. BRCA1 and BARD1 were quantified from immunoblots (graph right).
D Immunoprecipitated BARD1 complexes were combined with E1, E2 (UBE2D1), Ub and ATP. The complexes purified with WT-BARD1 exhibited ligase activity whereas R99E-BARD1 and L44R-BARD1 precipitated complexes had minimal activity with Ub conjugation components.
E Comparison of heterodimer bearing R99E-BARD1 or I26A- or C61G –BRCA1 to catalyse the formation of Ub chains in vitro. Ub mix refers to E1, E2, Ub, ATP and ligase reaction buffer.
Supplementary Figure 4 BRCA1–BARD1 ligase activity promotes DNA resection in the presence of 53BP1.
A Intensity and the area of RPA foci in EdU positive cells treated with BARD1 siRNA and complemented with siRNA resistant WT, R99E- or L44R-BARD1 variants (n= 60 cells, bars= S.E. *= p<0.05, ***= p<0.005 Student t-test, compared to WT). Quantified from data shown in Fig 4a.
B Camptothecin sensitivity (2.5uM) of cells treated with BARD1 siRNA and complemented with siRNA resistant WT or R99E BARD1. Restoration of resistance requires CtIP (3 replicates per experiment, 3 experiments, error bars = S.E. *= p<0.05, Student t-test).
C DR3-GFP substrate and repair products are illustrated left, BRCA1 or BARD1 depletion reduce gene conversion of a substrate integrated into U20S cells after I-Sce-1 transfection, (n=5, 3 technical repeats per experiment, bars = S.E. ***= p<0.005, Student t-test).
D Gene conversion in cells treated with BARD1 siRNA and complemented with WT or R99E-BARD1. Restoration of gene conversion by 53BP1 depletion requires CtIP. (n= 4, assays, each assay 3 technical repeats, bars = S.E. ***= p<0.005, Student t-test).
E 53BP1 depletion increases RPA foci in cells complemented with R99E-BARD1. Cells treated with BARD1 siRNA and complemented with WT-BARD1 or R99E-BARD1, with or without treatment with 53BP1 siRNA. Cells were fixed 2 hours post 5 Gy IR. EdU positive cells were scored for the number of RPA foci. n=>30 cells, bars = S.E. ***= p<0.005, Student t-test.
F As in E, stained for RAD51.
Supplementary Figure 5 53BP1 effector proteins counter BRCA1–BARD1 ligase activity in olaparib and camptothecin sensitivity.
A Artemis depletion promotes resistance of R99E-BARD1 complemented cells and BARD1 depleted cells to Camptothecin, Olaparib and IR. Cells were treated with siRNAs shown plated and clones counted 10-14 days later. Colony numbers are expressed as % of untreated cells. (3 replicates per experiment, 3 experiments, error bars = S.E. *= p<0.05, *** p<0.005, ns: not significant).
B As in A but following Rev7 depletion.
C Lysates from cells treated with siRNAs shown, subject to SDS-PAGE and immunoblotted with the antibodies shown.
Supplementary Figure 6 An H2A-Ub fusion promotes DNA resection, HR and resistance to olaparib and camptothecin.
A H2A-C-terminal K to R mutant had no impact on Olaparib sensitivity in colony survival assays. H2A and H2A mutants were expressed for 72 hours prior to Olaparib exposure (10 μm) n=3, replicates per experiment 3, error bars = S.E.
B Incorporation of H2A WT and H2A-mutants into chromatin. Sol = soluble fraction (100 mM salt), Nuc= nuclear fraction (200 mM salt) and Pel= Pellet (500 mM salt).
C Western blot for protein levels following BARD1 and 53BP1 depletions and H2A and H2A-Ub expressions for experiments shown in Supplemental Fig. 6D & F).
D H2A-Ub expression restores gene conversion of an integrated substrate. DR3-GFP-U20S cells were transfected with the siRNAs shown and either WT H2A or H2A-Ub with I-Sce-1 before assessment of gene conversion (n= 3 assays, each assay 3 technical repeats, bars = S.E. ***= p<0.005, ns: not significant).
E Representative images of HA-H2A-Ub and RAD51 foci quantification (to accompany Fig 6C).
F RAD51 foci in EdU positive cells treated with BARD1, or BARD1 and 53BP1 siRNA and transfected with either WT H2A or H2A-Ub (n=30 cells, bars = S.E. ***= p<0.005.).
G H2A-Ub, but not H2A or H2A-BFP expression improves survival of BARD1 depleted cells after Olaparib and Camptothecin exposure. Cells were induced with for H2A expression and treated with BARD1 or control siRNA for 72 hours before exposure to Olaparib (10 μM) or Camptothecin (2.5 μM). n=3, replicates per experiment 3, error bars = S.E. *= p<0.05, Students T test, ns: not significant.
H H2A-Ub expression has no impact on cellular sensitivity of BARD1 depleted cells to Hydroxyurea. Inducible cells were doxycycline and siRNA treated as above and exposed to 3mM Hydroxyurea for 16 hours then plated and clones counted 14 days later. 3 replicates per experiment, 3 experiments, error bars = S.E. *= p<0.05, Students T test.
I Western blot for protein levels following BARD1 depletion and H2A, H2A-Ub or H2A-BFP expressions for experiments shown in Supplemental Fig. 6G&H.
J Restoration of Olaparib (10 μM) and Camptothecin (2.5 μM) resistance of BARD1 depleted cells by H2A-Ub requires CtIP. Inducible H2A-expressing cells were doxycycline and siRNA treated as above and exposed to the agents shown, plated and colonies counted 14 days later. n=>3, replicates per experiment 3, error bars = S.E. *= p<0.05, Students T test.
K RAD51 foci quantification in EdU+ cells depleted for BARD1 and expressing H2A-Ub after co-depletion of BARD1 or BARD1 and CtIP. (n=3, 50 cells per replicate, error bars = S.E. *= p<0.05, Students T test).
Supplementary Figure 7 Investigation of how BRCA1–BARD1 ligase activity or Ub-modified nucleosomes might affect 53BP1 and resection.
A Depletion of BRCA1 or BARD1 does not increase GFP expression from epigenetically silenced loci. Cells bearing integrated and epigenetically silenced CMV-GFP and EF-GFP were depleted as shown or treated with 1 mM Trichostatin A (TSA) histone deacetylase inhibitor. 3 replicates per experiment 3 experiments, error bars = S.E. *** p<0.005, Students T test, compared to NTC siRNA (left) or untreated control (right)).
B Expression of H2A-fusions does not increase GFP expression from epigenetically silenced loci.
C Depletion of CHD3 does not improve gene conversion in BARD1 depleted cells. Mean of 3 assays, each assay 3 technical repeats, bars = S.E. (knockdown shown in D).
D Depletion of CHD3 does not improve survival of BARD1 depleted cells treated with Olaparib (10 μM). Cells were treated with BARD1 siRNA or BARD1 and CHD3 siRNA for 72 hours before exposure to Olaparib, plated and colonies counted 10-14 days later. Colony numbers are expressed as % of untreated cells. (3 replicates per experiment 3 experiments, error bars = S.E.). Lysates from co-treated cells were immunoblotted for the proteins shown (right).
E C-terminal fusions of Ub to H2A do not inhibit the restoration of 53BP1 foci in MG132-treated cells mediated by N-terminal H2A Ub fusion. Cells were transfected with HA-H2A expression constructs Ub fusions. The HA-H2A was mutated at K13 15 118 119 and 125 127 129R and the fused Ub moiety carried K to R mutations in all lysines to prevent chain formation. Cells were treated with MG132 (10 μM) for 1 hour prior to irradiation (5Gy) and fixed with PFA prior to permeablization and stained for HA to identify H2A expressing cells, 53BP1 and γH2AX. HA-H2A expressing cells were scored for 53BP1 foci after 3 hours. (30 cells per mutant per technical repeat, 3 experiments repeats, bars =S.E. ** p<0.01, *** p<0.005, Students T test).
F Representative images of cell staining used in E.
G BLM accumulation to IRIF is reduced in cells depleted for BARD1. Cells were transfected with control or BARD1 siRNA, incubated for 72 hours before incubation with EdU for the last half hour, exposure to 5 Gy IR and further incubation for an hour before fixation and staining as shown. 100 cells per condition, 3 experimental repeats, bars =S.E *** p<0.005, Students T test.
Supplementary Figure 8 The nucleosome remodeler SMARCAD1 is in the pathway including BRCA1–BARD1 ligase activity.
A SMARCAD1 knock-down is epistatic with R99E-BARD1 in Camptothecin (2.5 μM) and Olaparib (10 μM) sensitivity. Colony numbers are expressed as % of untreated cells. (n=>3, replicates per experiment 3, error bars = S.E. * p<0.05, Student’s t test)
B SMARCAD1 knock-down is epistatic with R99E-BARD1 in gene conversion (mean of n>3 assays, each assay 3 technical repeats, bars = S.E., * p<0.05, ** p<0.05 Student’s t test).
C Rescue of RAD51 IRIF BARD1 depleted cells by H2A~Ub in requires SMARCAD1. SMARCAD1 is not needed in the absence of 53BP1. The graph below shows quantification. *** p<0.05, n>50 cells per repeat, 3 experimental repeats.
D The ATPase activity of SMARCAD1 influences 53BP1 positioning at IRIF. HeLa cells were transfected with siRNA targeting SMARCAD1 and incubated for 72 hours, exposed to 2 Gy IR and fixed 8 hours later, or transfected with siRNA targeting SMARCAD1 and with siRNA resistant plasmids for WT and ATPase mutant (K528R) myc-SMARCAD1 before treatment. Cells were immunostained for BRCA1 and 53BP1, imaged and deconvolved as in Fig 4D. 30 profiles measured over 3 experimental repeats. Bars =1 standard deviation.
E SMARCAD1 CUE domains, and ATPase activity promote resistance to Camptothecin (2.5 μM) and Olaparib (10 μM). Colony numbers are expressed as % of untreated cells (3 replicates per experiment, 3 experiments, error bars = S.E. * p<0.05).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Note (PDF 2760 kb)
Supplementary Data Set 1
Original blots for figures 1–7 (PDF 3051 kb)
Rights and permissions
About this article
Cite this article
Densham, R., Garvin, A., Stone, H. et al. Human BRCA1–BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection. Nat Struct Mol Biol 23, 647–655 (2016). https://doi.org/10.1038/nsmb.3236
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.3236
This article is cited by
-
HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage
Nature Structural & Molecular Biology (2023)
-
An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering
Nature Communications (2023)
-
Roles for the methyltransferase SETD8 in DNA damage repair
Clinical Epigenetics (2022)
-
BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1
Nature Communications (2022)
-
BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1
Nature Structural & Molecular Biology (2021)
