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.
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Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001)
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)
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)
Huen, M. S. et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 131, 901–914 (2007)
Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 131, 887–900 (2007)
Kolas, N. K. et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318, 1637–1640 (2007)
Doil, C. et al. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 136, 435–446 (2009)
Wang, B. et al. Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316, 1194–1198 (2007)
Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007)
Kim, H., Chen, J. & Yu, X. Ubiquitin-binding protein RAP80 mediates BRCA1-dependent DNA damage response. Science 316, 1202–1205 (2007)
Wu, J. et al. Histone ubiquitination associates with BRCA1-dependent DNA damage response. Mol. Cell. Biol. 29, 849–860 (2009)
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)
Nicassio, F. et al. Human USP3 is a chromatin modifier required for S phase progression and genome stability. Curr. Biol. 17, 1972–1977 (2007)
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)
Kayagaki, N. et al. DUBA: a deubiquitinase that regulates type I interferon production. Science 318, 1628–1632 (2007)
Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004)
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)
Edelmann, M. J. et al. Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem. J. 418, 379–390 (2009)
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)
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)
Natsume, T. et al. A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics. Anal. Chem. 74, 4725–4733 (2002)
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)
Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009)
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)
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)
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)
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)
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)
Galanty, Y. et al. Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. Nature 462, 935–939 (2009)
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)
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)
Hanna, J. et al. Deubiquitinating enzyme Ubp6 functions noncatalytically to delay proteasomal degradation. Cell 127, 99–111 (2006)
Quesada, V. et al. Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases. Biochem. Biophys. Res. Commun. 314, 54–62 (2004)
Nijman, S. M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005)
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)
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)
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)
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)
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.
The authors declare no competing financial interests.
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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
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