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


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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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.


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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.

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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.

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Correspondence to Shinichiro Nakada or Daniel Durocher.

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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).

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