Abstract

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14,5,6,7,8,9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

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Acknowledgements

We thank J. Young (Repare Therapeutics), M. Bashkurov (NBCC, LTRI), L. Kesselman (LTRI), S. Rossi (LTRI) and M. Rother (LUMC); and R. Greenberg (University of Pennsylvania) for the U2OS–FokI cells. We acknowledge that J. Lukas first proposed the name shieldin. S.M.N. is funded by a research fellowship from the Dutch Cancer Society (KWF). S.Ad. is a Banting post-doctoral fellow. D.S. is funded by a post-doctoral research fellowship from CIHR. H.v.A. is funded by the European Research Council (CoG-50364). T.H. is supported by MD Anderson Cancer Center Support Grant P30 CA016672 and the Cancer Prevention Research Institute of Texas (CPRIT/RR160032). Work in the A.M. laboratory is funded through CIHR grant PJT153307. A.-C.G. is funded by CIHR grant FDN143301. Work in the C.J.L. laboratory is funded by Programme Grants from Cancer Research UK (CRUK/A14276) and Breast Cancer Now (CTR-Q4-Y2). Work in the S.R. and J.J. laboratories is funded by the Dutch Cancer Society (KWF 2014-6532), the Netherlands Organization for Scientific Research (VICI 91814643 and a National Roadmap grant for Large-Scale Research Facilities to J.J.), the Swiss National Science Foundation (310030_156869 to S.R.), the European Research Council (CoG-681572 to S.R. and SyG-319661 to J.J.). D.D. and A.C.-G. are Canada Research Chairs (Tier I). D.D. is funded by CIHR grant FDN143343, Canadian Cancer Society (CCS grant #705644) and OICR grant OICR-OC-TRI.

Author information

Author notes

  1. These authors contributed equally: Sylvie M. Noordermeer, Salomé Adam, Dheva Setiaputra

Affiliations

  1. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    • Sylvie M. Noordermeer
    • , Salomé Adam
    • , Dheva Setiaputra
    • , Michele Olivieri
    • , Alejandro Álvarez-Quilón
    • , Nathalie Moatti
    • , Michal Zimmermann
    • , Alana Sherker
    • , Sébastien Landry
    • , Rachel K. Szilard
    • , Meagan M. Munro
    • , Andrea McEwan
    • , Théo Goullet de Rugy
    • , Zhen-Yuan Lin
    • , Anne-Claude Gingras
    •  & Daniel Durocher
  2. Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

    • Sylvie M. Noordermeer
    •  & Haico van Attikum
  3. Division of Molecular Pathology, Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands

    • Marco Barazas
    • , Stefano Annunziato
    • , Jos Jonkers
    •  & Sven Rottenberg
  4. The CRUK Gene Function Laboratory and Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London, UK

    • Stephen J. Pettitt
    • , Dragomir B. Krastev
    • , Feifei Song
    • , Inger Brandsma
    • , Jessica Frankum
    • , Rachel Brough
    •  & Christopher J. Lord
  5. Department of Immunology, University of Toronto, Toronto, Ontario, Canada

    • Alexanda K. Ling
    •  & Alberto Martin
  6. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    • Michele Olivieri
    • , Alana Sherker
    • , Jason Moffat
    • , Anne-Claude Gingras
    •  & Daniel Durocher
  7. Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

    • Traver Hart
  8. Donnelly Centre, University of Toronto, Toronto, Ontario, Canada

    • Jason Moffat
  9. Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland

    • Sven Rottenberg

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Contributions

S.M.N. initiated the project in the D.D. laboratory, performed the RPE1 BRCA1KO screen, validated hits and performed functional follow up. S.Ad. performed validation and localization studies. D.S. carried out structure-function analyses and DNA binding studies. M.B. performed mouse tumour model studies. S.J.P. analysed the SUM149PT talazoparib screen and validated hits. A.K.L. generated knockouts in CH12F3-2 cells, carried out class switch recombination assays and analysed AID levels. M.O. performed the ionizing radiation screen and validation. A.A.-Q. carried out immunopurifications and helped with immunofluorescence quantification. N.M. performed class switch recombination, traffic light reporter and RPA phosphorylation assays. M.Z. carried out the SUM149PT olaparib screen and generated the RPE1 BRCA1KO cells. S.An. examined Shld1 and Shld2 mutations in mouse embryonic stem cells. D.B.K., I.B., F.S., J.F. and R.B. performed the SUM149PT talazoparib screen and validation. I.B. and F.S. developed SUM149PT knockout cells. F.S. carried out SUM149PT epistasis experiments. A.S. performed the REV7 IP-MS. S.L. initiated work on REV7. R.K.S. generated vectors and edited the manuscript. M.M.M. generated IP-MS cell lines. A.Mc. carried out screen analysis, cloning and TIDE. T.G.d.R. performed shieldin qPCR. Z.-Y.L. carried out IP-MS for SHLD1–3 under supervision of A.-C.G. T.H. analysed screen data and built the TKOv2 library with J.M. A.Ma. supervised A.K.L. J.J. and S.R. supervised M.B. and S.An. D.D. and H.v.A. supervised S.M.N. C.J.L. supervised S.J.P., D.B.K., F.S., I.B., J.F. and R.B. S.M.N. and D.D. wrote the manuscript with input from the other authors.

Competing interests

D.D. and T.H. are advisors to Repare Therapeutics. C.J.L. is a named inventor on patents describing the use of PARP inhibitors (DK3044221 (T3), ES2611504 (T3), US2014378525 (A1), WO2008020180 (A2), WO2009027650 (A1)) and stands to gain from their use as part of the ICR ‘Rewards to Inventors’ scheme.

Corresponding author

Correspondence to Daniel Durocher.

Extended data figures and tables

  1. Extended Data Fig. 1 The identification of the shieldin complex and its role in the response to genotoxic treatments.

    a, Schematic of the PARPi resistance screens. b, Competitive growth assays determining the capacity of the indicated sgRNAs to cause resistance to PARP inhibitors in RPE1 BRCA1KO cells. Data are presented as the mean fraction of GFP-positive cells ± s.e.m., normalized to day 0 (n = 3, independent viral transductions). Gene-editing efficiencies of the sgRNAs can be found in Supplementary Table 2. Note that we have not been able to obtain TIDE data for the ATMIN-targeting sgRNAs. c, Representative images of SUM149PT–Cas9 cells transfected with indicated crRNAs (see Methods) and exposed to 50 nM talazoparib for 14 d. Purple colour indicates cells detected by Incucyte live-cell imaging. Scale bar, 100 μm. The data are a representative set of images from two biologically independent experiments. d, Screenshot of the genomic locus surrounding human CTC-534A2.2 taken from ENSEMBL. e, Schematic of the screen performed in RPE1-hTERT TP53−/− cells stably expressing Cas9 to study genes mediating ionizing radiation-sensitivity. f, g, Competitive growth assays measuring the capacity of the indicated sgRNAs to cause resistance to etoposide (100 nM) in RPE1 wild-type cells (f) or PARPi (16 nM) in RPE1 BRCA1KO cells (g). Data are presented as the mean fraction of GFP-positive cells ± s.d., normalized to day 0 (n = 3, independent viral transductions). Gene-editing efficiencies of the sgRNAs can be found in Supplementary Table 2. h, Talazoparib sensitivity in 11 SHLD1KO SUM149PT clones obtained after co-transfection of tracrRNA and one of four distinct SHLD1 crRNAs (5-1, 5-2, 5-3 or 5-5). Each clone was exposed to talazoparib in a 384-well plate format for 5 days. As a comparison, talazoparib sensitivity in parental SUM149PT cells with wild-type SHLD1 (WT) is shown, as is talazoparib resistance in a BRCA1 revertant subclone (BRCA1-rev) of SUM149PT50. Bars represent the mean ± s.d. (n = 4 biologically independent experiments). ANOVA was performed for each SHLD1KO clone versus wild type using Dunnett correction for multiple comparisons, P < 1015. Gene-editing efficiencies can be found in Supplementary Table 2. i, BRCA1KO and BRCA1KOSHLD2KO cells were virally transduced with expression vectors for GFP alone or GFP–SHLD2. Sensitivity to olaparib (200 nM) was determined by a short-term survival assay in the presence of 1 μg ml−1 doxycycline to induce protein expression. Data are represented as dots for every individual experiment with the bar representing the mean ± s.d. (n = 3). Source Data

  2. Extended Data Fig. 2 Shieldin inhibits homologous recombination.

    a, Representative micrographs of RAD51 focus formation in the indicated RPE1 cell lines (data quantified in Fig. 2d, n ≥ 3). b, Traffic light reporter assay testing RPE1 BRCA1KO cells virally transduced with sgRNAs targeting 53BP1 or SHLD3. Data are represented as dots for individual experiments with the bar representing the mean ± s.d. (n = 3). Gene-editing efficiencies of the sgRNAs can be found in Supplementary Table 2. c, Representative flow cytometry plots of cells analysed with the traffic light reporter assay (data quantified in Fig. 2e, n ≥ 3). d, Representative flow cytometry plots of cells analysed with the traffic light reporter assay (data quantified in b). Source Data

  3. Extended Data Fig. 3 Mouse shieldin promotes resistance to PARP inhibition in Brca1-mutated cells and tumours.

    a, Clonogenic survival assays of transduced KB1P-G3 cells treated with indicated olaparib doses ± ATM inhibitor (ATMi) KU60019 (500 nM). On day 6, the ATMi alone and untreated groups were stopped and stained with 0.1% crystal violet; the other groups were stopped and stained on day 9. Data shown are representative of 3 biologically independent experiments (with 3 technical replicates each). b, Left, quantification of RAD51 focus formation in parental KB1P-G3 (Brca1/;Trp53/) cells or KB1P-G3 cells that were transduced with the indicated lentiviral sgRNA vectors. Cells were fixed without treatment or 4 h after irradiation (10-Gy dose). Each data point represents a microscopy field containing a minimum of 50 cells; the bar represents the mean ± s.d. (n = 15). Right, representative micrographs of RAD51-negative and RAD51-positive cells (the latter is indicated by an arrowhead). DNA was stained with DAPI. c, Clonogenic survival assay of Rosa26CreERT2/wt;Brca1Δ/Δ;p53-null mouse embryonic stem cells virally transduced with the indicated sgRNA and treated without or with 15 nM olaparib for 7 d. Gene-editing efficiencies of the sgRNAs can be found in Supplementary Table 2. Data shown are representative of 3 biologically independent experiments (with ≥ 2 technical replicates each). d, Clonogenic survival assay of Rosa26CreERT2/wt;Brca1Sco/Δ mouse embryonic stem cells virally transduced with the indicated sgRNA and treated without or with 0.5 µM tamoxifen to induce BRCA1 depletion. Gene-editing efficiencies of the sgRNAs can be found in Supplementary Table 2. Data shown are representative of 2 biologically independent experiments (with 3 technical replicates each). Source Data

  4. Extended Data Fig. 4 Shieldin localizes to DSB sites.

    a, Representative micrographs of the experiments quantified in Fig. 3c. b, Representative micrographs of the experiments quantified in Fig. 3e. c, Quantification of mRNAs for SHLD1, SHLD2 and SHLD3. RPE1 cells were transfected with  siCTRL (non-targeting control siRNA) or siRNA targeting the indicated shieldin subunits. Forty-eight hours after transfection, mRNA was purified and reverse-transcribed before being assayed by quantitative real-time PCR. Data were normalized to the amount of GAPDH mRNA and expressed relative to the corresponding value for cells transfected with siCTRL. Data are presented as the mean ± s.d. (n = 3, independent siRNA transfections). d, Whole cell extracts from RPE1 wild-type cells transfected with the indicated siRNAs were processed for immunoblotting with the indicated antibodies. Tubulin is used as a loading control (n = 1 experiment; siRNA efficiency is also monitored by immunofluorescence). e, Quantification of 53BP1 and RIF1 recruitment to ionizing radiation-induced DSBs (1 h after irradiation with 10 Gy) following depletion of the indicated shieldin components. Data are represented as the mean ± s.d. (n = 3, independent siRNA transfections). f, Representative micrographs of the experiments quantified in e. Source Data

  5. Extended Data Fig. 5 Epistasis between 53BP1 and shieldin factors.

    a, Quantification of RAD51 focus formation 3 h after irradiation (10 Gy) in RPE1 BRCA1KO (left), BRCA1KO53BP1KO (middle) and BRCA1KOSHLD2KO (right) cells after viral transduction with the indicated sgRNAs (editing efficiency can be found in Supplementary Table 2) or empty vector (EV). Data are represented as the mean ± s.d. (for BRCA1KO53BP1KO, n = 4 biologically independent immunofluorescence experiments; for BRCA1KO and BRCA1KOSHLD2KO, n = 6 biologically independent immunofluorescence experiments). P values were calculated using a two-tailed unpaired t-test. Left, BRCA1KO EV versus sg53BP1-1 P = 0.0002; EV versus sgSHLD1-1 P = 0.0043; EV versus sgSHLD2-2 P = 0.0348; EV versus sgSHLD3-1 P = 0.0180; EV versus sgREV7-1 P = 0.0012). Middle, right: all comparisons to the EV condition were non-significant (NS). Values for BRCA1KO53BP1KO EV versus sg53BP1-1 P = 0.2332; EV versus sgSHLD1-1 P = 0.4451; EV versus sgSHLD2-2 P = 0.9632; EV versus sgSHLD3-1 P = 0.1187; EV versus sgREV7-1 P = 0.0568. Values for BRCA1KOSHLD2KO: EV versus sg53BP1-1 P = 0.0550; EV versus sgSHLD1-1 P = 0.1864; EV versus sgSHLD2-2 P = 0.3568; EV versus sgSHLD3-1 P = 0.4641; EV versus sgREV7-1 P = 0.2888. b, Talazoparib sensitivity of wild type or two independent SHLD1KO SUM149PT-dox-Cas9 clones (A and D) virally transduced with an sgRNA targeting 53BP1 (sg53BP1) or a control non-targeting sgRNA (sgCtrl), following induction of Cas9. Data are presented as the mean ± s.d. (n = 3 biologically independent experiments). Source Data

  6. Extended Data Fig. 6 The co-localization of shieldin with RIF1 on chromatin.

    a, Representation of the deletion mutants of SHLD2-N used in c, d. The orange shading indicates blocks of homology. b, Schematic of the LacR–RIF1 chromatin recruitment assay. c, Quantification of the experiment shown in d. Colocalization was considered positive when the average GFP intensity at the mCherry focus was threefold over background nuclear intensity. A minimum of 20 cells were imaged per biological replicate (circles); the bar represents the mean ± s.d. (n = 3). d, Representative images of the data quantified in c. The main focus is shown in inset ; scale bar, 10 μm. eh, Quantification (e, g) and representative micrographs (f, h) of overexpressed GFP–SHLD2-N and mCherry–LacR–RIF1(1–967) co-transfected into uninduced U2OS–FokI cells along with siRNA against shieldin complex subunits after processing for mCherry and GFP (e, f) or mCherry and REV7 (g, h) immunofluorescence. Colocalization was considered positive when the average GFP or REV7 intensity at the mCherry focus was threefold over background nuclear intensity. A minimum of 20 cells were imaged per condition (circles); the bar represents the mean ± s.d. (n = 3 biologically independent experiments). i, Representative images of the data quantified in j. The main focus is shown in inset; scale bar, 10 μm. j, Quantification of GFP intensity at the mCherry–LacR–RIF1(1–967) focus, normalized to nuclear background. Each data point represents a cell transfected with the vector coding for the indicated GFP fusion. The line is at the median. The data are an aggregate of three independent experiments with a minimum of 20 cells counted (total cells counted: 62, 60 and 61 for GFP, GFP–SHLD2-C and GFP–SHLD3, respectively). k, mCherry–LacR–FokI colocalization with full-length or N-terminally truncated (Δ1–50) GFP–SHLD2. Mean normalized focus intensity is shown from a total of 59 (full-length SHLD2) or 56 (SHLD2 Δ1–50) cells counted (n = 2 biologically independent experiments). Source Data

  7. Extended Data Fig. 7 Mapping the architecture of the shieldin complex.

    a, Streptavidin pulldown analysis determining which region of SHLD2 associates with the other shieldin subunits. WCEs of 293T cells transfected with an expression vectors for Flag–SHLD1, V5–SHLD3, GFP–REV7 and Strep/HA-tagged SHLD2, SHLD2-N (residues 2–420), SHLD2-C (residues 421–904) or empty Strep/HA vector (EV) were incubated with streptavidin resin and bound proteins were eluted with biotin. WCEs and elutions were analysed by SDS–PAGE and immunoblotting with the indicated antibodies. Tubulin was used as a loading control. Results are representative set of immunoblots from two independent experiments. Asterisk denotes a non-specific band. b, Mapping the SHLD3 and REV7 binding sites on the SHLD2 N terminus through streptavidin pulldowns with different SHLD2 constructs (detailed in Extended Data Fig. 6a) and immunoblotting. Results are a representative of a set of immunoblots from three independent experiments. c, Affinity purification of shieldin complex components using N-terminally truncated SHLD2 (Δ1–50) analysed by immunoblotting (representative of three independent experiments). d, Streptavidin pulldown analysis of SHLD2 association with REV7 and SHLD3. 293T cells were transfected with siRNAs and expression vectors for epitope-tagged shieldin components as indicated (EV, empty Strep/HA vector). WCEs were incubated with streptavidin resin and bound proteins were eluted with biotin. WCEs and elutions were analysed by SDS–PAGE and immunoblotted with the indicated antibodies. Short and long exposures are shown for GFP and V5 immunoblots (n = 1). e, Dependency of V5–SHLD3 co-immunoprecipitation with GFP–REV7. 293T cells were transfected with siRNAs and expression vectors for epitope-tagged REV7 and SHLD3 as indicated (EV, empty V5 vector). WCEs were incubated with anti-V5 antibody and protein G resin. Bound proteins were boiled in SDS sample buffer and analysed by immunoblotting with GFP and V5 antibodies (n = 1). f, Association between SHLD3 and RIF1. WCEs of 293T cells transfected with an expression vector for unfused GFP (−) or GFP–SHLD3 (SHLD3) were incubated with GFP-Trap resin. Bound proteins were boiled in SDS sample buffer and analysed by SDS–PAGE and immunoblotting against 53BP1 and RIF1. Results are representative of 2 SHLD3 immunoprecipitations, using SHLD3 fused to GFP (shown here) and V5 (shown in Fig. 3g) affinity tags.

  8. Extended Data Fig. 8 Controls supporting the role of shieldin in promoting physiological NHEJ.

    a, Representative dot plots of the flow cytometry data obtained (of n = 3 biologically independent experiments) to assess class switching in Fig. 3h. Class switch recombination was determined as the percentage of IgA+ cells following stimulation after subtracting the baseline percentage of IgA+ cells in the indicated clones (values in parentheses). b, c, Epistasis analysis of shieldin and 53BP1 in class switch recombination. The percentage of class switching in CH12F3-2 wild type, single knockout or double knockout cells (as indicated) following stimulation is shown. Each data point represents a biological replicate; the line represents the mean ± s.d. (n = 3). Genomic editing efficiencies of the sgRNAs can be found in Supplementary Table 2. d, WCEs of the indicated CH12F2-3 clones were probed for AID and β-actin (loading control) by immunoblotting and were quantified by densitometry. Each data point represents a biological replicate; the line represents the mean ± s.d. (n = 9 for wild type, n = 3 for other samples). e, Random plasmid integration of linearized pcGFP-c1 conferring G418 resistance. Resistant colonies were quantified after 14 d. Bar represents the mean ± s.d. with wild-type cells set at 100% (left, n = 5; right, n = 4 except SHLD2KO (2.7) n = 3 biologically independent experiments). f, Representative images of the plasmid integration assays quantified in e. g, Un-irradiated CH12F3-2 clones were immunoblotted for RPA32 (also known as RPA2) phosphorylation (a representative set from n = 3 biological replicates; data relates to Fig. 3i). Source Data

  9. Extended Data Fig. 9 The role of DSB-targeted SHLD2 in the suppression of homologous recombination and the mapping of the SHLD2-C–SHLD1 complex binding to ssDNA.

    a, Representative micrographs of RPE1 BRCA1KO53BP1KO cells transduced with the indicated GFP-fusion proteins, pre-extracted, fixed and stained for RAD51 and GFP 3 h after ionizing radiation (10 Gy). Protein expression was induced for 24 h before exposure to ionizing radiation using 1 µg ml−1 doxycycline. Data relates to Fig. 4b. Note that owing to the pre-extraction required for visualization of RAD51 foci, the visualization of non-FHA-tagged SHLD2 is lost. b, SDS–PAGE analysis of purified SHLD2-C–SHLD1 complexes. Strep/HA–SHLD2(421–904)–Flag-SHLD1 complexes were purified from transiently transfected 293T cells. Concentrations of purified proteins were estimated by Coomassie staining and comparison to a standard curve of known BSA concentrations visualized by fluorescence at 700 nm. SHLD2-C m1 and SHLD2(S)-C denote SHLD2-C constructs carrying the OB-fold m1 mutation and the internal deletion (Δ655–723) corresponding to the naturally occurring splice variant of SHLD2, respectively. Open and filled arrowheads mark the bands corresponding to SHLD2-C and SHLD1, respectively. EV refers to empty Strep/HA vector. A representative stained gel from two independent experiments is shown. c, Representative image of the 32P-labelled ssDNA EMSA with SHLD2-C–SHLD1 for Kd determination shown in Fig. 4e. d, Model of the SHLD2 OB-fold domains and the engineered mutations (red spheres, point mutations; red ribbons, splice variant deletion). Model relates to Fig. 4b, d.

  10. Extended Data Fig. 10 SHLD2 OB-folds are required for suppression of RAD51 focus formation induced by ionizing radiation.

    a, Quantification of RAD51 foci 3 h after 10 Gy irradiation in RPE1 BRCA1KOSHLD2KO cells complemented with the indicated GFP-tagged SHLD2 constructs via viral transduction. Protein expression was induced with 1 µg ml−1 doxycycline for 24 h before exposure to ionizing radiation. Each data point is a biological replicate; the bar represents the mean ± s.d. (n = 6 for BRCA1KO untransduced cells, BRCA1KOSHLD2KO untransduced and GFP-SHLD2 cells, n = 3 for remaining cell lines, biologically independent experiments). b, Representative micrographs of the data shown in a. Note that owing to the pre-extraction required for visualization of RAD51 foci, the visualization of non-FHA tagged SHLD2 foci is lost. c, Representative micrographs of RPE1 BRCA1KOSHLD2KO cells virally transduced with vectors expressing GFP-tagged SHLD2 wild type or its OB-fold m1 mutant (m1), or short splice variant (S), 1 h after 5 Gy ionizing radiation. Scale bar, 10 μm. d, Quantification of the data shown in c. Each data point represents an independent biological replicate counting ≥ 100 cells. Data are represented as mean ± s.d. (n = 3). e, WCEs of 293T cells co-transfected with Strep/HA–SHLD2 wild type, Strep/HA–SHLD2 m1 or Strep/HA–SHLD2(S) mutants, and other shieldin subunits (Flag–SHLD1, V5–SHLD3, and GFP–REV7) were incubated with streptavidin resin and bound proteins were eluted with biotin. WCEs and eluted proteins were visualized by SDS–PAGE and immunoblotting with the indicated antibodies. Results shown are a representative set from two independent experiments. Source Data

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Note 1: Shieldin recruitment data. An overview of the experiments conducted to determine the recruitment hierarchy of the Shieldin complex.

  2. Reporting Summary

  3. Supplementary Figure 1

    This file contains an overview of all uncropped Western blots presented in the main and extended data figures.

  4. Supplementary Table 1

    Gene-based results of PARPi resistance screens. Gene-based enrichment scores as calculated using MaGeCK (see methods section for details). The output of MaGECK MLE module is as follows: sgRNA; the number of targeting gRNAs; beta score, which is similar to the log2 fold change; z-score of the beta value; P-value; FDR; the P-value using the Wald-test (wald-p-value) and the FDR using the Wald test (wald-FDR). Results per cell line and drug are represented in the different tabs.

  5. Supplementary Table 2

    Genotype information for all gene-editing experiments. Mutational spectrum of clonal CRISPR/Cas9 edited cell lines (tab 1) and gene editing efficiencies of pooled sgRNA transductions and transfections (tab 2) as determined by genomic sequencing and TIDE analysis.

  6. Supplementary Table 3

    This file contains the proteins identified in the different bait-based IP-MS experiments (see methods section for details). The first column indicates the used bait for IP; the second and third columns indicate the identified interactor; the fourth column displays the average spectral count of the prey (averaged over the different replicates); the fifth column displays the BFDR as calculated by SAINTexpress.

  7. Supplementary Table 4

    Gene-based results of IR sensitivity screen. Gene-based results as calculated using DrugZ (see methods section for details). The output of the DrugZ algorithm includes sumZ score which is the sum of each guide-level Z-score for a gene; The ‘numObs’, a count of the number of guides per gene multiplied by the number of replicates; The normZ, a normalized sumZ score; the P-value for depleted genes; the rank of depleted genes and the FDR for depleted genes. The next three columns represent the P-value, rank and FDR for enriched genes.

  8. Supplementary Table 5

    An overview of the sgRNA sequences used in this study (human sgRNAs: tab 1; mouse sgRNAs: tab 2).

  9. Supplementary Table 6

    An overview of the genomic PCR primers used in this study to check editing of sgRNAs (human genomic primers: tab 1; mouse genomic primers: tab 2).

  10. Supplementary Table 7

    Antibodies used in this project. An overview of the antibodies used in this project, including supplier information and dilutions used for the different applications.

Source data

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DOI

https://doi.org/10.1038/s41586-018-0340-7

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