Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1

Abstract

DNA double-strand breaks (DSBs) pose a potent threat to genome integrity. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DSBs elicit a signalling cascade that modifies the chromatin surrounding the break, first by ATM-dependent phosphorylation and then by RNF8-, RNF168- and BRCA1-dependent regulatory ubiquitination. Here we report that OTUB1, a deubiquitinating enzyme, is an inhibitor of DSB-induced chromatin ubiquitination. Surprisingly, we found that OTUB1 suppresses RNF168-dependent poly-ubiquitination independently of its catalytic activity. OTUB1 does so by binding to and inhibiting UBC13 (also known as UBE2N), the cognate E2 enzyme for RNF168. This unusual mode of regulation is unlikely to be limited to UBC13 because analysis of OTUB1-associated proteins revealed that OTUB1 binds to E2s of the UBE2D and UBE2E subfamilies. Finally, OTUB1 depletion mitigates the DSB repair defect associated with defective ATM signalling, indicating that pharmacological targeting of the OTUB1–UBC13 interaction might enhance the DNA damage response.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: OTUB1 modulates ubiquitination of chromatin after DNA damage.
Figure 2: OTUB1 inhibits the DSB response downstream of RNF168 focal accumulation.
Figure 3: OTUB1 interacts with UBC13 to inhibit ubiquitin chain formation.
Figure 4: OTUB1 directly inhibits UBC13.
Figure 5: Inhibition of OTUB1 rescues ATM inhibition.

References

  1. 1

    Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001)

    CAS  Article  Google Scholar 

  2. 2

    Stewart, G. S. et al. RIDDLE immunodeficiency syndrome is linked to defects in 53BP1-mediated DNA damage signaling. Proc. Natl Acad. Sci. USA 104, 16910–16915 (2007)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Stewart, G. S. et al. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 136, 420–434 (2009)

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    ADS  CAS  Article  Google Scholar 

  7. 7

    Doil, C. et al. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 136, 435–446 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Wang, B. et al. Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316, 1194–1198 (2007)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Kim, H., Chen, J. & Yu, X. Ubiquitin-binding protein RAP80 mediates BRCA1-dependent DNA damage response. Science 316, 1202–1205 (2007)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Wu, J. et al. Histone ubiquitination associates with BRCA1-dependent DNA damage response. Mol. Cell. Biol. 29, 849–860 (2009)

    CAS  Article  Google Scholar 

  12. 12

    Komander, D., Clague, M. J. & Urbé, S. Breaking the chains: structure and function of the deubiquitinases. Nature Rev. Mol. Cell Biol. 10, 550–563 (2009)

    CAS  Article  Google Scholar 

  13. 13

    Nicassio, F. et al. Human USP3 is a chromatin modifier required for S phase progression and genome stability. Curr. Biol. 17, 1972–1977 (2007)

    CAS  Article  Google Scholar 

  14. 14

    Shao, G. et al. The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks. Proc. Natl Acad. Sci. USA 106, 3166–3171 (2009)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Kayagaki, N. et al. DUBA: a deubiquitinase that regulates type I interferon production. Science 318, 1628–1632 (2007)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Tran, H., Hamada, F., Schwarz-Romond, T. & Bienz, M. Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains. Genes Dev. 22, 528–542 (2008)

    CAS  Article  Google Scholar 

  18. 18

    Edelmann, M. J. et al. Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem. J. 418, 379–390 (2009)

    CAS  Article  Google Scholar 

  19. 19

    Wang, T. et al. Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1. J. Mol. Biol. 386, 1011–1023 (2009)

    CAS  Article  Google Scholar 

  20. 20

    Balakirev, M. Y., Tcherniuk, S. O., Jaquinod, M. & Chroboczek, J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 4, 517–522 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Natsume, T. et al. A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics. Anal. Chem. 74, 4725–4733 (2002)

    CAS  Article  Google Scholar 

  22. 22

    Goudreault, M. et al. A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein. Mol. Cell. Proteomics 8, 157–171 (2009)

    CAS  Article  Google Scholar 

  23. 23

    Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M. & Wolberger, C. Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nature Struct. Mol. Biol. 13, 915–920 (2006)

    CAS  Article  Google Scholar 

  25. 25

    Petroski, M. D. et al. Substrate modification with lysine 63-linked ubiquitin chains through the UBC13-UEV1A ubiquitin-conjugating enzyme. J. Biol. Chem. 282, 29936–29945 (2007)

    CAS  Article  Google Scholar 

  26. 26

    VanDemark, A. P., Hofmann, R. M., Tsui, C., Pickart, C. M. & Wolberger, C. Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell 105, 711–720 (2001)

    CAS  Article  Google Scholar 

  27. 27

    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)

    CAS  Article  Google Scholar 

  28. 28

    Rappold, I., Iwabuchi, K., Date, T. & Chen, J. Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways. J. Cell Biol. 153, 613–620 (2001)

    CAS  Article  Google Scholar 

  29. 29

    Galanty, Y. et al. Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. Nature 462, 935–939 (2009)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Pierce, A. J., Hu, P., Han, M., Ellis, N. & Jasin, M. Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev. 15, 3237–3242 (2001)

    CAS  Article  Google Scholar 

  31. 31

    Beucher, A. et al. ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2. EMBO J. 28, 3413–3427 (2009)

    CAS  Article  Google Scholar 

  32. 32

    Hanna, J. et al. Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127, 99–111 (2006)

    CAS  Article  Google Scholar 

  33. 33

    Quesada, V. et al. Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases. Biochem. Biophys. Res. Commun. 314, 54–62 (2004)

    CAS  Article  Google Scholar 

  34. 34

    Nijman, S. M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005)

    CAS  Article  Google Scholar 

  35. 35

    Kittler, R., Heninger, A. K., Franke, K., Habermann, B. & Buchholz, F. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells. Nature Methods 2, 779–784 (2005)

    CAS  Article  Google Scholar 

  36. 36

    Méndez, J. & Stillman, B. Chromatin association of human origin recognition complex, Cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000)

    Article  Google Scholar 

  37. 37

    Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002)

    CAS  Article  Google Scholar 

  38. 38

    Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658 (2003)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to T. Ikura, S. Olivarius, R. Sakasai, A. Shibata, T. Oikawa, J. Unno, Y. Katuski, I. Imoto, S. Koyasu, R. Greenberg and the Core Instrumentation Facility, Keio University School of Medicine for technical support and reagents. We also thank R. Szilard and S. Angers for reading the manuscript. Work in the Nakada group in the Suda laboratory is supported by the Promotion of Environmental Improvement for Independence of Young Researchers, ‘Kanrinmaru Project’ from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan, Grant-in-Aid for Young Scientists (Startup 2009, 21870033) from MEXT, Takeda Science Foundation, Mitsubishi Pharma Research Foundation and the Daiwa Anglo-Japanese Foundation. I.T. is supported by MEXT’s Distinctive University Education Support Program ‘Good Practice’. Work in the Natsume laboratory is supported by the New Energy and Industrial Technology Development Organization (NEDO). S.P. holds a studentship from the Boehringer Ingelheim Fonds. D.D. is the Thomas Kierans Chair in Mechanisms of Cancer Development and a Canada Research Chair (Tier 2) in Proteomics, Bioinformatics and Functional genomics. Work in the Durocher and Gingras laboratories is supported by grants MOP10703115 (DD) and MOP84314 (ACG) from the Canadian Institutes of Health Research.

Author information

Affiliations

Authors

Contributions

S.N. and D.D. designed the experiments. S.N. performed most of the experiments described in the text. I.T. performed several experiments. M.M. and S.P. generated the inducible Flag–OTUB1 cell line. S.P. performed immunofluorescence for UbK63, RNF168 and BRCA1 and carried out the ‘Toronto’ immunoprecipitation-tandem mass spectrometry (IP-MS/MS). L.O. carried out the DR-GFP assays. Y.-C.J. carried out binding experiments with charged E2s and ubiquitination assays with UBE2D2 and UBE2L3. A.A.-H. carried out RNF168 immunoprecipitations. F.S. supervised Y.-C.J.; A.-C.G. analysed the ‘Toronto’ IP-MS/MS data. S.-i.I. and T.N. performed and analysed the ‘Tokyo’ IP-MS/MS. A.K. constructed plasmids and purified recombinant proteins. T.S. supervised I.T.; S.N. and D.D. wrote the manuscript.

Corresponding authors

Correspondence to Shinichiro Nakada or Daniel Durocher.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-18 with legends and Supplementary Tables 1-3. (PDF 5401 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Nakada, S., Tai, I., Panier, S. et al. Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1. Nature 466, 941–946 (2010). https://doi.org/10.1038/nature09297

Download citation

Further reading

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.