Letter | Published:

53BP1–RIF1–shieldin counteracts DSB resection through CST- and Polα-dependent fill-in

Naturevolume 560pages112116 (2018) | Download Citation

Abstract

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair—which requires a 3′ overhang—and classical non-homologous end joining, which can join unresected ends1,2. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1)3,4,5,6,7,8. When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7–SHLD1–SHLD2–SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors4,6,7,8,9. Here we address the mechanism by which 53BP1–RIF1–shieldin regulates the generation of recombinogenic 3′ overhangs. We report that CTC1–STN1–TEN1 (CST)10, a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)–primase11, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST–Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

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

References

  1. 1.

    Zimmermann, M. & de Lange, T. 53BP1: pro choice in DNA repair. Trends Cell Biol. 24, 108–117 (2014).

  2. 2.

    Panier, S. & Boulton, S. J. Double-strand break repair: 53BP1 comes into focus. Nat. Rev. Mol. Cell Biol. 15, 7–18 (2014).

  3. 3.

    Bouwman, P. et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat. Struct. Mol. Biol. 17, 688–695 (2010).

  4. 4.

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

  5. 5.

    Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).

  6. 6.

    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).

  7. 7.

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

  8. 8.

    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).

  9. 9.

    Noordermeer, S. M. et al. The shieldin complex mediates 53BP1-dependent DNA repair. Nature https://doi.org/10.1038/s41586-018-0340-7 (2018).

  10. 10.

    Price, C. M. et al. Evolution of CST function in telomere maintenance. Cell Cycle 9, 3177–3185 (2010).

  11. 11.

    Casteel, D. E. et al. A DNA polymerase-α·primase cofactor with homology to replication protein A-32 regulates DNA replication in mammalian cells. J. Biol. Chem. 284, 5807–5818 (2009).

  12. 12.

    Griffith, J. D. et al. Mammalian telomeres end in a large duplex loop. Cell 97, 503–514 (1999).

  13. 13.

    Doksani, Y., Wu, J. Y., de Lange, T. & Zhuang, X. Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation. Cell 155, 345–356 (2013).

  14. 14.

    Palm, W. & de Lange, T. How shelterin protects mammalian telomeres. Annu. Rev. Genet. 42, 301–334 (2008).

  15. 15.

    Lazzerini-Denchi, E. & Sfeir, A. Stop pulling my strings–what telomeres taught us about the DNA damage response. Nat. Rev. Mol. Cell Biol. 17, 364–378 (2016).

  16. 16.

    Wu, P., Takai, H. & de Lange, T. Telomeric 3′ overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST. Cell 150, 39–52 (2012).

  17. 17.

    Goulian, M., Heard, C. J. & Grimm, S. L. Purification and properties of an accessory protein for DNA polymerase α/primase. J. Biol. Chem. 265, 13221–13230 (1990).

  18. 18.

    Sfeir, A. & de Lange, T. Removal of shelterin reveals the telomere end-protection problem. Science 336, 593–597 (2012).

  19. 19.

    Lottersberger, F., Bothmer, A., Robbiani, D. F., Nussenzweig, M. C. & de Lange, T. Role of 53BP1 oligomerization in regulating double-strand break repair. Proc. Natl Acad. Sci. USA 110, 2146–2151 (2013).

  20. 20.

    Kibe, T., Zimmermann, M. & de Lange, T. TPP1 blocks an ATR-mediated resection mechanism at telomeres. Mol. Cell 61, 236–246 (2016).

  21. 21.

    Feng, X., Hsu, S. J., Kasbek, C., Chaiken, M. & Price, C. M. CTC1-mediated C-strand fill-in is an essential step in telomere length maintenance. Nucleic Acids Res. 45, 4281–4293 (2017).

  22. 22.

    Gu, P. et al. CTC1 deletion results in defective telomere replication, leading to catastrophic telomere loss and stem cell exhaustion. EMBO J. 31, 2309–2321 (2012).

  23. 23.

    Lottersberger, F., Karssemeijer, R. A., Dimitrova, N. & de Lange, T. 53BP1 and the LINC complex promote microtubule-dependent DSB mobility and DNA repair. Cell 163, 880–893 (2015).

  24. 24.

    van Steensel, B., Smogorzewska, A. & de Lange, T. TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413 (1998).

  25. 25.

    Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. & de Lange, T. p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283, 1321–1325 (1999).

  26. 26.

    Celli, G. B. & de Lange, T. DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nat. Cell Biol. 7, 712–718 (2005).

  27. 27.

    Hockemeyer, D., Daniels, J. P., Takai, H. & de Lange, T. Recent expansion of the telomeric complex in rodents: two distinct POT1 proteins protect mouse telomeres. Cell 126, 63–77 (2006).

  28. 28.

    Takai, H. et al. A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus. Genes Dev. 30, 812–826 (2016).

  29. 29.

    Hom, R. A. & Wuttke, D. S. Human CST prefers G-rich but not necessarily telomeric sequences. Biochemistry 56, 4210–4218 (2017).

  30. 30.

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

  31. 31.

    Xu, X. et al. Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol. Cell 3, 389–395 (1999).

  32. 32.

    Frank, K. M. et al. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396, 173–177 (1998).

  33. 33.

    Kibe, T., Osawa, G. A., Keegan, C. E. & de Lange, T. Telomere protection by TPP1 is mediated by POT1a and POT1b. Mol. Cell. Biol. 30, 1059–1066 (2010).

  34. 34.

    Tang, J. et al. Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. Nat. Struct. Mol. Biol. 20, 317–325 (2013).

  35. 35.

    Gong, Y., Handa, N., Kowalczykowski, S. C. & de Lange, T. PHF11 promotes DSB resection, ATR signaling, and HR. Genes Dev. 31, 46–58 (2017).

  36. 36.

    Takai, H., Wang, R. C., Takai, K. K., Yang, H. & de Lange, T. Tel2 regulates the stability of PI3K-related protein kinases. Cell 131, 1248–1259 (2007).

  37. 37.

    Mirzoeva, O. K. & Petrini, J. H. DNA damage-dependent nuclear dynamics of the Mre11 complex. Mol. Cell. Biol. 21, 281–288 (2001).

Download references

Acknowledgements

We thank D. White for mouse husbandry; N. Bosco, R. Karssemeijer, L. Timashev and Y. Doksani for help with CRISPR gene knockouts, image analysis and generating MEFs; and R. Greenberg and C. Price for providing cell lines. The Rockefeller University BioImaging Center provided assistance. This work was supported by grants from the NCI (R35CA210036), ACS and BCRF to T.d.L., a grant from the CIHR (FDN143343) to D.D. and the Banting Postdoctoral fellowship to M.Z.

Author information

Author notes

    • Francisca Lottersberger

    Present address: Department of Medical and Health Sciences, Linköping University, Linköping, Sweden

  1. These authors contributed equally: Zachary Mirman, Francisca Lottersberger

Affiliations

  1. Laboratory for Cell Biology and Genetics, Rockefeller University, New York, NY, USA

    • Zachary Mirman
    • , Francisca Lottersberger
    • , Hiroyuki Takai
    • , Tatsuya Kibe
    • , Yi Gong
    • , Kaori Takai
    • , Alessandro Bianchi
    • , Michal Zimmermann
    •  & Titia de Lange
  2. Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK

    • Alessandro Bianchi
  3. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada

    • Michal Zimmermann
    •  & Daniel Durocher

Authors

  1. Search for Zachary Mirman in:

  2. Search for Francisca Lottersberger in:

  3. Search for Hiroyuki Takai in:

  4. Search for Tatsuya Kibe in:

  5. Search for Yi Gong in:

  6. Search for Kaori Takai in:

  7. Search for Alessandro Bianchi in:

  8. Search for Michal Zimmermann in:

  9. Search for Daniel Durocher in:

  10. Search for Titia de Lange in:

Contributions

M.Z. initiated this work in the de Lange laboratory. T.K. and F.L. performed resection assays. Z.M. and H.T. performed CST and Polα localization assays and RPA phosphorylation assays. Z.M. performed PARPi and RAD51 assays. Y.G. and T.K. analysed RPA foci. A.B. performed yeast two-hybrid assays. K.T. and H.T. performed co-immunoprecipitation analysis. K.T. and T.d.L. performed telomere fusion assays. D.D. provided information, reagents and advice. T.d.L. conceived the study and wrote the paper with input from all co-authors.

Competing interests

D.D. is a founder of, owns equity in and receives funding from Repare Therapeutics.

Corresponding author

Correspondence to Titia de Lange.

Extended data figures and tables

  1. Extended Data Fig. 1 Shieldin and CST counteract telomere hyper-resection.

    a-c, Effect of SHLD2 on hyper-resection at telomeres that lack TPP1. a, Immunoblot for CHK1-P, an indicator of TPP1 deletion, in Tpp1f/f MEFs with and without bulk population treatment with a Shld2 sgRNA and/or Cre (representative of three experiments). b, Quantitative analysis of telomere end resection as in Fig. 1c using the cells shown in a. c, Quantification of the extent of resection detected in b, as in Fig. 1d. Mean (centre bars) and s.d. (error bars) from four independent experiments. *P < 0.05, **P < 0.01, two-tailed Welch’s t-test. d, Fluorescence-activated cell sorting (FACS) profiles of the indicated cells incubated with BrdU to measure S phase effects of the Stn1 shRNA. Gating strategy for live cells and singlets is shown below the FACS profiles. Representative of two experiments. e, f, Experiments to verify that the single-stranded DNA signal derives from a 3′ overhang. e, Immunoblot for STN1 and γ-tubulin in Tpp1f/f (Rif1f/+) cells treated with Stn1 shRNA and/or Cre. Representative of two experiments. f, Quantitative assay for telomeric overhangs, as in Fig. 1c. Plugs in the ExoI lanes were treated with the 3′ exonuclease from E. coli. Representative of two experiments.

  2. Extended Data Fig. 2 Hyper-resection at telomeres that lack TPP1 is counteracted by CST and shieldin.

    a, Immunoblots showing absence of REV7 and reduction of STN1 expression in the indicated Tpp1f/f and Tpp1f/fRev7−/− MEFs treated with either Ctc1 or Stn1 shRNA. Diminished STN1 expression is used as a proxy for the efficacy of the Ctc1 shRNA. Representative of two experiments. b, Quantitative analysis of telomeric overhangs, as in Fig. 1c. Representative of two experiments. c, Quantification of the effect of Ctc1 and Stn1 shRNA on resection at telomeres that lack TPP1, as in Fig. 1d. Data are obtained from two independent REV7-proficient and two independent REV7-deficient clones (light and dark shading).

  3. Extended Data Fig. 3 No effect of CST depletion on telomere hyper-resection when 53BP1 or RIF1 are absent.

    a, SV40LT-immortalized Tpp1f/f53bp1−/− cells were complemented with wild-type 53BP1 or a mutant 53BP1 that lacks the ability to interact with RIF1, treated with a Stn1 shRNA as indicated and analysed by immunoblotting for 53BP1 and STN1. Representative of four experiments. b, Quantitative analysis of telomeric overhangs, as in Fig. 1c. c, Quantification of the resection at telomeres that lack TPP1, in four independent experiments performed as in Fig. 1d. d, Immunoblots showing loss of RIF1 and STN1 in the indicated Tpp1f/fRif1f/+ and Tpp1f/fRif1f/f MEFs treated with Cre (96 h) as indicated, and with or without Stn1 shRNA. Note the diminished levels of RIF1 after Cre, owing to heterozygosity in the Tpp1f/fRif1f/+ cells. e, Quantitative analysis of telomeric overhangs, as in Fig. 1c. f, Quantification of the extent of resection detected, as in e, determined from three independent experiments (indicated by different shades of grey) showing mean (centre bars) and s.d. (error bars). Each experiment involved all indicated samples analysed in parallel. g, h, Experiments to verify that the single-stranded DNA signal derives from a 3′ overhang. g, Immunoblot for STN1 and γ-tubulin in Tpp1f/fRif1f/f cells treated with Stn1 shRNA and/or Cre. Representative of two experiments. h, Quantitative assay for telomeric overhangs, as in Fig. 1c. Plugs in the ExoI lanes were treated with the 3′ exonuclease from E. coli. Representative of two experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Welch's t-test.

  4. Extended Data Fig. 4 SHLD2 counteracts resection at telomeres that lack TRF2.

    a, Immunoblots for TRF2 deletion and CHK2 phosphorylation in Trf2f/fLig4−/− MEFs, with and without bulk population treatment with a Shld2 sgRNA and/or Cre. Asterisk, non-specific band. Representative of three experiments. b, Quantitative analysis of telomere end resection, as in Fig. 1c, using the cells shown in a. c, Quantification of the extent of resection detected in b, as in Fig. 1d. Mean (centre bars) and s.d. (error bars) from four independent experiments. *P < 0.05, two-tailed Welch's t-test.

  5. Extended Data Fig. 5 CST interacts with shieldin.

    a, Immunoprecipitation of individual mouse CST subunits or the three subunit complex (each subunit bearing a Myc tag) with Flag-tagged mouse SHLD1, co-expressed in 293T cells. Flag-tagged POT1B and POT1A serve as positive and negative controls for CST binding, respectively. Representative of two experiments. b, Two-hybrid analysis of CST-shieldin interaction. Yeast cultures were grown overnight in synthetic complete medium that lacked tryptophan and leucine, to a density of 5 × 107 cells per millilitre. Serial tenfold dilutions were generated and 4 μl of each dilution was spotted on synthetic complete medium that lacked the nutrients tryptophan, leucine, adenine and histidine, and contained 3-aminotriazole (3-AT), as indicated. Plates were then incubated for 5 days at 30 °C before imaging. Representative of three experiments.

  6. Extended Data Fig. 6 Localization of CST and Polα to DSBs.

    a, Quantification of HA–STN1 localization to DSBs induced by FOKI, as in Fig. 3e. Mean (centre bars) and s.d. (error bars) from 4–6 independent experiments, with >80 induced nuclei for each condition in each experiment. b, Immunofluorescence for endogenous Polα in FOKI–LacI U2OS cells in S phase and after RO3306 treatment (G2). Dotted lines denote the outline of the nucleus. Representative of two experiments. c, Examples of HA–STN1 and Polα localization at DSBs induced by FOKI in G2-arrested FOKI–LacI U2OS cells (as in Fig. 3f). Representative of three experiments. d, Quantification of co-localization of Polα with DSBs induced by FOKI (as in Fig. 3f). Mean (centre bars) and s.d. (error bars) from three independent experiments, with >80 induced nuclei for each condition in each experiment. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Welch’s t-test.

  7. Extended Data Fig. 7 Effect of STN1 knockdown on the intensity of RPA foci induced by ionizing radiation.

    Quantification of Myc–RPA32 intensity per nucleus in the experiments shown in Fig. 3g, h. Medians (centre bars and numbers below) obtained from four independent experiments, with >20 nuclei for each experimental condition in each experiment. Each symbol represents one nucleus. *P < 0.05, ****P < 0.0001, two-tailed Welch's t-test.

  8. Extended Data Fig. 8 Effect of CST and Polα on PARPi treatment of BRCA1-deficient cells.

    af, Immunoblots on the MEFs used in Fig. 4a–e to verify the absence of deleted proteins and efficacy of the shRNAs. Reduction in STN1 expression is used as a proxy for the efficacy of the Ctc1 shRNA because no antibody to mouse CTC1 is available. Each immunoblot is representative of three experiments. g, Immunoblots for BRCA1 and STN1 in the cells used in Fig. 4f. Representative of two experiments. hj, Control experiment to assess that cells analysed in Fig. 4f progressed through S phase during treatment with PARPi. h, Experimental timeline, as in Fig. 4f, but with inclusion of BrdU in the medium during treatment with PARPi. i, Example of the assay for the presence of BrdU (immunofluorescence) in metaphases collected after the experimental timeline, as in h. j, Quantification of the BrdU incorporation into metaphase chromosomes, as in i (one experiment with ten metaphases per condition).

Supplementary information

  1. Supplementary Figure 1

    This file contains the uncropped blots.

  2. Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41586-018-0324-7

Further reading

  • 1.

    The shieldin complex mediates 53BP1-dependent DNA repair

    • Sylvie M. Noordermeer
    • , Salomé Adam
    • , Dheva Setiaputra
    • , Marco Barazas
    • , Stephen J. Pettitt
    • , Alexanda K. Ling
    • , Michele Olivieri
    • , Alejandro Álvarez-Quilón
    • , Nathalie Moatti
    • , Michal Zimmermann
    • , Stefano Annunziato
    • , Dragomir B. Krastev
    • , Feifei Song
    • , Inger Brandsma
    • , Jessica Frankum
    • , Rachel Brough
    • , Alana Sherker
    • , Sébastien Landry
    • , Rachel K. Szilard
    • , Meagan M. Munro
    • , Andrea McEwan
    • , Théo Goullet de Rugy
    • , Zhen-Yuan Lin
    • , Traver Hart
    • , Jason Moffat
    • , Anne-Claude Gingras
    • , Alberto Martin
    • , Haico van Attikum
    • , Jos Jonkers
    • , Christopher J. Lord
    • , Sven Rottenberg
    •  & Daniel Durocher

    Nature (2018)

  • 2.

    Assembling a protective shield

    • Roger A Greenberg

    Nature Cell Biology (2018)

  • 3.

    An OB-fold complex controls the repair pathways for DNA double-strand breaks

    • Shengxian Gao
    • , Sumin Feng
    • , Shaokai Ning
    • , Jingyan Liu
    • , Huayu Zhao
    • , Yixi Xu
    • , Jinfeng Shang
    • , Kejiao Li
    • , Qing Li
    • , Rong Guo
    •  & Dongyi Xu

    Nature Communications (2018)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.