Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

53BP1 nuclear bodies enforce replication timing at under-replicated DNA to limit heritable DNA damage

Nature Cell Biology volume 21, pages 487–497 (2019)Cite this article

Subjects

Abstract

Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown. Here, we show that the formation of 53BP1-NBs interrupts the chain of iterative damage intrinsically embedded in UR-DNA. Unlike clastogen-induced 53BP1 foci that are repaired throughout interphase, 53BP1-NBs restrain replication of the embedded genomic loci until late S phase, thus enabling the dedicated RAD52-mediated repair of UR-DNA lesions. The absence or malfunction of 53BP1-NBs causes premature replication of the affected loci, accompanied by genotoxic RAD51-mediated recombination. Thus, through adjusting replication timing and repair pathway choice at under-replicated loci, 53BP1-NBs enable the completion of genome duplication of inherited UR-DNA and prevent the conversion of stochastic under-replications into genome instability.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Dissolution of 53BP1-NBs is coupled to local DNA replication in late S phase.
Fig. 2: 53BP1-NBs confine replication of UR-DNA to late S phase.
Fig. 3: RIF1 enforces late replication timing at 53BP1-marked UR-DNA.
Fig. 4: RAD52 catalyses the replication-coupled repair of inherited UR-DNA.
Fig. 5: Loss of 53BP1 from UR-DNA triggers chromosome segregation defects.
Fig. 6: Unresolved UR-DNA produces pathological recombination intermediates.

Similar content being viewed by others

Data availability

Numerical and statistical source data for Figs. 1b,c,f,g, 2c, 3b,c,e, 4a,c,d, 5b,d,e, 6a and Supplementary Figs. 1c,d,h, 2a, 4a,f, 5a,e, and 6a,b,d,f,g have been provided in Supplementary Table 1. Primary image data of fixed cells (Figs. 1e, 3d, 4a, 5b,c, 6a and Supplementary Figs. 2a, 3a, and 4b,c,e; 3D maximum intensity projection, 24-bit colour overlay images in TIF format) and uncropped movies with their corresponding single, full-size images of time-series data for single-cell tracking experiments data (Figs. 1d, 2a,b, 3f, 4b and Supplementary Figs. 1e–g, 3b,c, and 5c; AVI movies and 3D maximum intensity projection, 24-bit colour overlay images in TIF format) have been deposited at the European Bioinformatics Institute (EBI) BioStudies database (https://www.ebi.ac.uk/biostudies/) with accession number S-BSST227. Also deposited under the same accession number at the BioStudies database are image source data (uncropped movies and corresponding single, full-size images of time-series data as 3D maximum intensity projection, 24-bit colour overlay images in TIF format) used to generate data for Figs. 1c,f,g, 2c, 3b,e, 4c and Supplementary Figs. 1h, 4a, 5a, and 6a,b). Source data acquired by QIBC (Figs. 1b, 3c, 4a,d, 5b,d,e, 6a and Supplementary Figs. 1c,d, 2a, 4f, 5e, and 6d,f,g), including software guidance to navigate image data, are available from the corresponding authors upon reasonable request.

References

  1. Tomasetti, C. & Vogelstein, B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 347, 78–81 (2015).

    Article  CAS  Google Scholar 

  2. Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    Article  CAS  Google Scholar 

  3. Somyajit, K. et al. Redox-sensitive alteration of replisome architecture safeguards genome integrity. Science 358, 797–802 (2017).

    Article  CAS  Google Scholar 

  4. Vujanovic, M. et al. Replication fork slowing and reversal upon DNA damage require PCNA polyubiquitination and ZRANB3 DNA translocase activity. Mol. Cell 67, 882–890 (2017).

    Article  CAS  Google Scholar 

  5. Al Mamun, M. et al. Inevitability and containment of replication errors for eukaryotic genome lengths spanning megabase to gigabase. Proc. Natl Acad. Sci. USA 113, E5765–E5774 (2016).

    Article  CAS  Google Scholar 

  6. Glover, T. W., Wilson, T. E. & Arlt, M. F. Fragile sites in cancer: more than meets the eye. Nat. Rev. Cancer 17, 489–501 (2017).

    Article  CAS  Google Scholar 

  7. Bergoglio, V. et al. DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis. J. Cell Biol. 201, 395–408 (2013).

    Article  CAS  Google Scholar 

  8. Minocherhomji, S. et al. Replication stress activates DNA repair synthesis in mitosis. Nature 528, 286–290 (2015).

    Article  CAS  Google Scholar 

  9. Chan, K. L., North, P. S. & Hickson, I. D. BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges. EMBO J. 26, 3397–3409 (2007).

    Article  CAS  Google Scholar 

  10. Chan, K. L., Palmai-Pallag, T., Ying, S. & Hickson, I. D. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat. Cell Biol. 11, 753–760 (2009).

    Article  CAS  Google Scholar 

  11. Baumann, C., Körner, R., Hofmann, K. & Nigg, E. A. PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint. Cell 128, 101–114 (2007).

    Article  CAS  Google Scholar 

  12. Naim, V. & Rosselli, F. The FANC pathway and BLM collaborate during mitosis to prevent micro-nucleation and chromosome abnormalities. Nat. Cell Biol. 11, 761–768 (2009).

    Article  CAS  Google Scholar 

  13. Harrigan, J. A. et al. Replication stress induces 53BP1-containing OPT domains in G1 cells. J. Cell Biol. 193, 97–108 (2011).

    Article  CAS  Google Scholar 

  14. Lukas, C. et al. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat. Cell Biol. 13, 243–253 (2011).

    Article  CAS  Google Scholar 

  15. Feng, W. & Jasin, M. BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination. Nat. Commun. 8, 1117 (2017).

    Article  Google Scholar 

  16. Martín-Pardillos, A. et al. Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions. Blood 130, 1523–1534 (2017).

    Article  Google Scholar 

  17. Flach, J. et al. Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells. Nature 512, 198–202 (2014).

    Article  CAS  Google Scholar 

  18. Arora, M., Moser, J., Phadke, H., Basha, A. A. & Spencer, S. L. Endogenous replication stress in mother cells leads to quiescence of daughter cells. Cell Rep. 19, 1351–1364 (2017).

    Article  CAS  Google Scholar 

  19. Lezaja, A. & Altmeyer, M. Inherited DNA lesions determine G1 duration in the next cell cycle. Cell Cycle 17, 24–32 (2018).

    Article  CAS  Google Scholar 

  20. Yang, H. W., Chung, M., Kudo, T. & Meyer, T. Competing memories of mitogen and p53 signalling control cell-cycle entry. Nature 549, 404–408 (2017).

    Article  CAS  Google Scholar 

  21. Burrell, R. A. et al. Replication stress links structural and numerical cancer chromosomal instability. Nature 494, 492–496 (2013).

    Article  CAS  Google Scholar 

  22. S Pedersen, R. et al. Profiling DNA damage response following mitotic perturbations. Nat. Commun. 7, 13887 (2016).

    Article  CAS  Google Scholar 

  23. Leonhardt, H. et al. Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271–280 (2000).

    Article  CAS  Google Scholar 

  24. Cornacchia, D. et al. Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells. EMBO J. 31, 3678–3690 (2012).

    Article  CAS  Google Scholar 

  25. Toledo, L. I. et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 (2013).

    Article  CAS  Google Scholar 

  26. Dilley, R. L. et al. Break-induced telomere synthesis underlies alternative telomere maintenance. Nature 539, 54–58 (2016).

    Article  CAS  Google Scholar 

  27. Polo, S. E. & Jackson, S. P. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev. 25, 409–433 (2011).

    Article  CAS  Google Scholar 

  28. Huen, M. S. Y. et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 131, 901–914 (2007).

    Article  CAS  Google Scholar 

  29. Kolas, N. K. et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318, 1637–1640 (2007).

    Article  CAS  Google Scholar 

  30. Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 131, 887–900 (2007).

    Article  CAS  Google Scholar 

  31. Huang, J. et al. RAD18 transmits DNA damage signalling to elicit homologous recombination repair. Nat. Cell Biol. 11, 592–603 (2009).

    Article  CAS  Google Scholar 

  32. Watanabe, K. et al. RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1. Nucleic Acids Res. 37, 2176–2193 (2009).

    Article  CAS  Google Scholar 

  33. 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  CAS  Google Scholar 

  34. Chapman, J. R. et al. RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression of DNA double-strand break resection. Mol. Cell 49, 858–871 (2013).

    Article  CAS  Google Scholar 

  35. Di Virgilio, M. et al. Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching. Science 339, 711–715 (2013).

    Article  Google Scholar 

  36. 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).

    Article  Google Scholar 

  37. Foti, R. et al. Nuclear architecture organized by RIF1 underpins the replication-timing program. Mol. Cell 61, 260–273 (2016).

    Article  CAS  Google Scholar 

  38. Zimmermann, M., Lottersberger, F., Buonomo, S. B., Sfeir, A. & de Lange, T. 53BP1 regulates DSB repair using Rif1 to control 5′ end resection. Science 339, 700–704 (2013).

    Article  CAS  Google Scholar 

  39. Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2016).

    Article  Google Scholar 

  40. Alver, R. C., Chadha, G. S., Gillespie, P. J. & Blow, J. J. Reversal of DDK-mediated MCM phosphorylation by RIF1-PP1 regulates replication initiation and replisome stability independently of ATR/Chk1. Cell Rep. 18, 2508–2520 (2017).

    Article  CAS  Google Scholar 

  41. Hiraga, S. I. et al. Rif1 controls DNA replication by directing protein phosphatase 1 to reverse Cdc7-mediatedphosphorylation of the MCM complex. Genes Dev. 28, 372–383 (2014).

    Article  CAS  Google Scholar 

  42. Dave, A., Cooley, C., Garg, M. & Bianchi, A. Protein phosphatase 1 recruitment by Rif1 regulates DNA replication origin firing by counteracting DDK activity. Cell Rep. 7, 53–61 (2014).

    Article  CAS  Google Scholar 

  43. Moreno, A. et al. Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells. Proc. Natl Acad. Sci. USA 113, E5757–E5764 (2016).

    Article  CAS  Google Scholar 

  44. Gupta, R. et al. DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity. Cell 173, 972–988 (2018).

    Article  CAS  Google Scholar 

  45. Tomida, J. et al. FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells. EMBO J. 37, e99543 (2018).

    Article  Google Scholar 

  46. Dev, H. et al. Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells. Nat. Cell Biol. 20, 954–965 (2018).

    Article  CAS  Google Scholar 

  47. Mirman, Z. et al. 53BP1–RIF1–shieldin counteracts DSB resection through CST- and Polα-dependent fill-in. Nature 560, 112–116 (2018).

    Article  CAS  Google Scholar 

  48. Noordermeer, S. M. et al. The shieldin complex mediates 53BP1-dependent DNA repair. Nature 560, 117–121 (2018).

    Article  CAS  Google Scholar 

  49. Ghezraoui, H. et al. 53BP1 cooperation with the REV7–shieldin complex underpins DNA structure-specific NHEJ. Nature 560, 122–127 (2018).

    Article  CAS  Google Scholar 

  50. Ochs, F. et al. 53BP1 fosters fidelity of homology-directed DNA repair. Nat. Struct. Mol. Biol. 23, 714–721 (2016).

    Article  CAS  Google Scholar 

  51. Boersma, V. et al. MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end resection. Nature 521, 537–540 (2015).

    Article  CAS  Google Scholar 

  52. Xu, G. et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature 521, 541–544 (2015).

    Article  CAS  Google Scholar 

  53. Feng, Z. et al. Rad52 inactivation is synthetically lethal with BRCA2 deficiency. Proc. Natl Acad. Sci. USA 108, 686–691 (2011).

    Article  CAS  Google Scholar 

  54. Tiwari, A., Addis Jones, O. & Chan, K. L. 53BP1 can limit sister-chromatid rupture and rearrangements driven by a distinct ultrafine DNA bridging-breakage process. Nat. Commun. 9, 643 (2018).

    Article  Google Scholar 

  55. Johnson, R. D. & Jasin, M. Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells. EMBO J. 19, 3398–3407 (2000).

    Article  CAS  Google Scholar 

  56. Chan, Y. W., Fugger, K. & West, S. C. Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations. Nat. Cell Biol. 20, 92–103 (2018).

    Article  CAS  Google Scholar 

  57. Bakkenist, C. J. & Kastan, M. B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

    Article  CAS  Google Scholar 

  58. Kaidi, A. & Jackson, S. P. KAT5 tyrosine phosphorylation couples chromatin sensing to ATM signalling. Nature 498, 70–74 (2013).

    Article  CAS  Google Scholar 

  59. Brouwer, I. et al. Human RAD52 captures and holds DNA strands, increases DNA flexibility, and prevents melting of duplex DNA: implications for DNA recombination. Cell Rep. 18, 2845–2853 (2017).

    Article  CAS  Google Scholar 

  60. McIlwraith, M. J. & West, S. C. DNA repair synthesis facilitates RAD52-mediated second-end capture during DSB repair. Mol. Cell 29, 510–516 (2008).

    Article  CAS  Google Scholar 

  61. Lambert, S. et al. Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange. Mol. Cell 39, 346–359 (2010).

    Article  CAS  Google Scholar 

  62. Sotiriou, S. K. et al. Mammalian RAD52 functions in break-induced replication repair of collapsed DNA replication forks. Mol. Cell 64, 1127–1134 (2016).

    Article  CAS  Google Scholar 

  63. Bhowmick, R., Minocherhomji, S. & Hickson, I. D. RAD52 facilitates mitotic DNA synthesis following replication stress. Mol. Cell 64, 1117–1126 (2016).

    Article  CAS  Google Scholar 

  64. Mazina, O. M., Keskin, H., Hanamshet, K., Storici, F. & Mazin, A. V. Rad52 inverse strand exchange drives RNA-templated DNA double-strand break repair. Mol. Cell 67, 19–29 (2017).

    Article  CAS  Google Scholar 

  65. Keskin, K. et al. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436–439 (2014).

    Article  CAS  Google Scholar 

  66. Yasuhara, T. et al. Human Rad52 promotes XPG-mediated R-loop processing to initiate transcription-associated homologous recombination repair. Cell 175, 558–570 (2018).

    Article  CAS  Google Scholar 

  67. Shanbhag, N. M., Rafalska-Metcalf, I. U., Balane-Bolivar, C., Janicki, S. M. & Greenberg, R. A. ATM-dependent chromatin changes silence transcription in cis to DNA double-strand breaks. Cell 141, 970–981 (2010).

    Article  CAS  Google Scholar 

  68. Helmrich, A., Ballarino, M. & Tora, L. Collisions between replication and transcription complexes cause common fragile site instability at the longest human genes. Mol. Cell 44, 966–977 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Research funding in the Lukas Laboratory was provided by the Novo Nordisk Foundation (grant no. NNF14CC0001) and the Lundbeck Foundation (grant no. R264-2017-2819). The HeLa Kyoto cell line was a gift from S. Narumiya. The authors thank Y. Adachi, D. Durocher, R. Kanaar, C. Dinant, R. Strauss and S. Tateishi for sharing reagents for this study. They acknowledge G. Karemore, J. Bulkescher and J. Dreier (Protein Imaging Platform at Novo Nordisk Center for Protein Research) for technical support, and members of J. S. Hoffmann’s group and all members of the Lukas Laboratory for useful suggestions and critical comments on the manuscript.

Author information

Authors and Affiliations

  1. Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Julian Spies, Claudia Lukas, Kumar Somyajit, Maj-Britt Rask, Jiri Lukas & Kai John Neelsen

Authors
  1. Julian Spies
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claudia Lukas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kumar Somyajit
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maj-Britt Rask
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiri Lukas
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kai John Neelsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.S., J.L., C.L. and K.J.N. conceived the project. J.S. designed, performed and analysed all live cell experiments, analysed 53BP1-NB volumes and their PCNA colocalization using Imaris, performed confocal colocalization experiments of 53BP1 with EdU and PCNA, analysed BLM- and RPA-positive anaphase bridges, counted 53BP1-NBs in U2OS and hTERT-RPE1 cells by QIBC, performed western blotting experiments, and recorded all representative confocal immunofluorescene images. K.J.N. performed and analysed QIBC experiments to quantify 53BP1-NBs, measured and analysed the colocalization of 53BP1-NBs with RAD18, RAD51 or RAD52 using Imaris, and analysed SCEs in chromatin bridges. M.-B.R. and K.S. performed western blotting experiments. M.-B.R. and C.L. generated all cell lines used in the study. All authors interpreted data. J.S. designed the figures with the help of K.J.N., K.S. and C.L. K.J.N. and J.L. wrote the paper with the help of J.S. All authors commented on the manuscript.

Corresponding authors

Correspondence to Jiri Lukas or Kai John Neelsen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Replication-coupled dissolution of 53BP1-NBs.

a, Parental U2OS cells and a U2OS clone stably expressing RFP-53BP1 and GFP-PCNA were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. Uncropped Western blots in Supplementary Fig. 7. b, U2OS cells shown in Fig. 1b immunostained for endogenous PCNA. Cell cycle stages (G1 phase, 5 S-phase quintiles (I-V) and G2 phase) based on DAPI and PCNA intensities quantified by QIBC. Wide-field microscopy images (1-7) show representative PCNA patterns. The assay was performed 3 times with similar results. c, Number of endogenous 53BP1-NBs throughout interphase in U2OS and hTERT-RPE1 cells quantified by QIBC (also see b). Data represents mean ± SD (n = 3 independent experiments). Data points plotted over bars. d, U2OS cells immunostained for endogenous 53BP1 and PCNA and imaged by confocal microscopy. S phase staging based on PCNA patterns. See also Fig. 1e. e, Overlays of representative MIPs of U2OS cells stably expressing GFP-53BP1 and TagRFP PCNA monitored throughout interphase by time-lapse imaging. Scale bar, 12 µm. Arrow indicates a 53BP1-NB. The assay was performed twice with similar results. f, Left, representative MIP images of U2OS cells stably expressing RFP-53BP1 and GFP-PCNA monitored throughout interphase by time-lapse imaging. The outlined cell is analysed on the right. Scale bar, 12 µm. Right, volume of 53BP1-NBs (purple line), nuclear PCNA intensity (green line) and the colocalization of PCNA with 53BP1-NBs (Pearson’s coefficient, heatmap) of the cell on the left. Broken vertical lines mark period of highest PCNA-53BP1 colocalization. The assay was performed once. g, Representative MIP images of a cell analyzed in Fig. 1g. Arrow indicates a replication-coupled dissolution event in late S phase. Scale bar, 12 µm. h, Trajectories of individual 53BP1-NBs from U2OS cells stably expressing RFP-53BP1 and GFP-PCNA and pulsed with 0.2 µM Aphidicolin for 26 hours prior to imaging. Green and purple broken lines indicate colocalization of PCNA and 53BP1; end of the bar marks NB dissolution; vertical broken line separates early-mid from mid-late S phase. The assay was performed once and 51 NBs were analyzed to generate the graph. Statistical source data in Supplementary Table 1.

Supplementary Figure 2 RAD18-NBs form at UR-DNA independently of 53BP1 and reveal replication timing control by 53BP1.

a, Left panel, representative confocal MIP images of U2OS cells immunostained for endogenous 53BP1 and RAD18. Scale bar, 12 µm. Inset shows x 4 magnification of the indicated area. Right panel, U2OS cells stably expressing GFP-53BP1 were immunostained for GFP and endogenous RAD18, respectively. Colocalization frequencies of RAD18 with 53BP1-NBs were quantified by QIBC throughout interphase in fixed cells. Data represents mean ± SD (n = 3 independent experiments). Individual data points are plotted over bars. See Supplementary Fig. 1b for cell cycle staging. b, U2OS cells stably expressing RFP-53BP1 and GFP-RAD18 were analyzed by immunoblotting with indicated antibodies; loading control: NUDC. Asterisk indicates an unspecific band. The assay was performed twice with similar results. c, U2OS cells stably expressing GFP-RAD18 and TagRFP-PCNA were treated with control and 53BP1 siRNA for 4 days and analyzed by immunoblotting with indicated antibodies; loading control: KAP1. Asterisk indicates an unspecific band. The immunoblot was performed once. See Supplementary Fig. 7 for uncropped Western blots and Supplementary Table 1 for all statistical source data.

Supplementary Figure 3 Inactive 53BP1 triggers premature replication and NB dissolution defects.

a, Left Panel, U2OS cells stably expressing GFP-53BP1-MFFR and TagRFP-PCNA were depleted from endogenous 53BP1 for 4 days and immunoblotted with indicated antibodies. 53BP1-MFFR lacks the epitope detected by the anti-53BP1 antibody and is instead detected by an anti-GFP antibody; loading control: KAP1. The assay was performed twice with similar results. See Supplementary Fig. 7 for uncropped Western blots. Right panel, representative MIP images of cells as shown on the left immunostained with an anti-GFP antibody and an antibody recognizing the C-terminus of 53BP1, which is absent in the truncation mutant. Scale bar, 12 µm. The immunoblot was performed once. b, Representative MIP images acquired by confocal imaging of a cell analyzed in Fig. 3b. Scale bar, 12 µm. Inset shows x 3 magnification of the indicated area. Arrow indicates a 53BP1-NB. The experiment was performed twice with similar results. c, Representative MIP images of S-phase cells from U2OS cells stably expressing GFP-53BP1-MFFR without depletion of endogenous 53BP1. Cells were treated with a control siRNA for 4 days prior to confocal microscopy. Arrows indicate two late dissolving 53BP1-MFFR-NBs. Scale bar, 12 µm. The assay was performed twice with similar results.

Supplementary Figure 4 Full-length 53BP1 regulates origin firing at 53BP1-NBs and rescues the dissolution defect of 53BP1-MFFR-NBs.

a, Trajectories of individual 53BP1-MFFR-NBs from cells treated as in Supplementary Fig. 3c. Green and purple broken lines indicate colocalization of PCNA and 53BP1-MFFR and the end of the bar marks the time of NB dissolution. Bars extending into G2 indicate undissolved 53BP1-NBs, vertical broken line separates early-mid from mid-late S phase. The assay was performed once and 51 NBs were analyzed to generate the graph. b, Representative MIP images of S-phase cells from U2OS cells stably expressing GFP-53BP1 and GFP-53BP1-MFFR immunostained for RIF1 and PCNA and imaged by confocal microscopy. Scale bar, 12 µm. Insets show x 4 magnification of the indicated areas. The assay was performed twice with similar results. c, Representative MIP images of U2OS cells stably expressing GFP-53BP1-MFFR, mock depleted or depleted of endogenous 53BP1 for 4 days, and immunostained for RIF1 and GFP. Cells were imaged by confocal microscopy. Scale bar, 12 µm. The assay was performed once. d, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted of endogenous 53BP1 and RIF1 for 4 days and 3 days, respectively, and immunoblotted with indicated antibodies; loading control: KAP1. The immunoblot was performed once. See Supplementary Fig. 7 for uncropped Western blots. e, Representative MIP images of U2OS cells stained for endogenous 53BP1, PCNA and 5-ethynyl-2´-deoxyuridine (EdU) as analyzed in f. Cells were imaged by confocal microscopy. Scale bar, 12 µm. The assay was performed twice with similar results. f, U2OS cells were mock treated or treated 3 hours with 20 µM CDC7 inhibitor prior to 15-minute pulse labelling with EdU and fixation. Cells were stained for endogenous 53BP1 and EdU and imaged by confocal microscopy. S phase staging was based on EdU patterns. The assay was independently performed twice with 355 and 193 cells analysed for replicate #1 and #2, respectively. g, U2OS cells were treated and stained as in f. EdU and DAPI intensities were measured by QIBC. The assay was performed twice with similar results. See Supplementary Table 1 for all statistical source data.

Supplementary Figure 5 RAD52 catalyzes replication-coupled repair of inherited UR-DNA.

a, Left panel, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted of endogenous 53BP1 and BRCA2 for 4 days and 2 days, respectively, and immunoblotted with indicated antibodies; loading control: KAP1. The assay was performed twice with similar results. Right panel, trajectories of individual 53BP1-NBs from U2OS cells depleted of endogenous 53BP1 and BRCA2 and monitored by confocal time-lapse imaging. Green and purple broken lines indicate colocalization of PCNA and 53BP1 and the end of the bar marks the time of NB dissolution, vertical broken line separates early-mid from mid-late S phase. 39 NBs from one experiment were analyzed to generate the bar graph. b, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted of endogenous 53BP1 and RAD52 for 4 days and 2 days, respectively, and immunoblotted with indicated antibodies; loading control: KAP1. The assay was performed twice with similar results. c, Representative MIP images acquired by confocal imaging of a cell analyzed in Fig. 4c. Scale bar, 12 µm. Arrow indicates a 53BP1-NB. The assay was performed twice with similar results. d, U2OS cells stably expressing GFP-53BP1 were treated with the indicated siRNAs for 3 days and immunoblotted with indicated antibodies; loading control: NUDC. The immunoblot was performed once. e, U2OS stably expressing GFP-53BP1 were treated with indicated siRNAs for 3 days prior to immunostaining for GFP and endogenous RAD52. Colocalization frequency of RAD52 with 53BP1-NBs was quantified by QIBC throughout interphase in fixed cells. Box and whisker plot with center line at median, box limit at 25th/75th centiles, whiskers indicate minimum and maximum, n = 6 independent experiments. Individual data points are plotted over boxes. See also d and Supplementary Fig. 1b for cell cycle staging. See Supplementary Fig. 7 for uncropped Western blots and Supplementary Table 1 for all numerical data.

Supplementary Figure 6 Loss of 53BP1 at UR-DNA triggers aberrant recombination events and chromosome segregation defects.

a, Left, 53BP1-NB trajectories from U2OS cells depleted of endogenous 53BP1 and REV7 and monitored by confocal time-lapse imaging. Green and purple broken lines indicate colocalization of PCNA and 53BP1 and the end of the bar marks NB dissolution. Bars extending into G2 indicate undissolved 53BP1-NBs, vertical broken line separates early-mid from mid-late S phase in a and b. 46 NBs from one experiment were analyzed to generate the graph. Right, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. b, Left, 53BP1-NB trajectories from U2OS cells depleted of endogenous 53BP1 and RINN1/SHLD3 and monitored by confocal time-lapse imaging. 40 NBs from 2 independent experiments were analyzed to generate the graph. Right, U2OS cells stably expressing RFP-53BP1 and GFP-PCNA were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. c, U2OS cells were depleted and immunoblotted with indicated siRNAs and antibodies; loading control: NUDC. The immunoblot was performed once. d, Frequency of SCE-negative chromatin bridges in U2OS cells in the first and second mitosis after treatment with 0.2 µM Aphidicolin (see Fig. 5a). Data represents mean ± SD (n = 3 independent experiments). e, HeLa cells depleted and immunoblotted with indicated siRNAs and antibodies; loading control: KAP1. The immunoblot was performed once. f, HeLa cells treated with 0.3 µM Aphidicolin as shown in Fig. 5a were immunostained for phospho-H3S10 and BrdU (also see e). Frequency of SCE-positive and SCE-negative chromatin bridges in the first and second mitosis after Aphidicolin treatment. Data represents mean ± SD (n = 4 independent experiments). g, HeLa cells treated with 0.2 µM Aphidicolin as shown in Fig. 5c were immunostained for FANCD2 and RPA (also see e). Frequency of FANCD2-positive and -negative anaphase bridges in the first and second mitosis after Aphidicolin treatment. Data represents mean ± SD (n = 3 independent experiments). Data points plotted over bars. Uncropped Western blots in Supplementary Fig. 7; statistical source data in Supplementary Table 1.

Supplementary Figure 7

Representative uncropped Western Blots.

Supplementary information

Supplementary Information

Supplementary Figures 1–7 and legend for Supplementary Table 1.

Reporting Summary

Supplementary Table 1

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Spies, J., Lukas, C., Somyajit, K. et al. 53BP1 nuclear bodies enforce replication timing at under-replicated DNA to limit heritable DNA damage. Nat Cell Biol 21, 487–497 (2019). https://doi.org/10.1038/s41556-019-0293-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-019-0293-6

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing