Systematic E2 screening reveals a UBE2D–RNF138–CtIP axis promoting DNA repair

Abstract

Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumorigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2 enzymes for impacts on cellular DSB responses. With a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3 ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.

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Figure 1: Screening E2s for IRIF kinetics.
Figure 2: Screening E2s for DSB repair.
Figure 3: Screening E2s for DDR signalling.
Figure 4: DDR validation of selected E2s.
Figure 5: DNA-end resection links UBE2Ds with RNF138.
Figure 6: Phenotypic mimicry between UBE2Ds and RNF138.
Figure 7: UBE2Ds- and RNF138-dependent CtIP accrual and its ionizing-radiation-induced ubiquitylation.
Figure 8: N-terminal, ionizing-radiation-induced CtIP ubiquitylation promotes its recruitment and function.

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Acknowledgements

We are grateful to all S.P.J. laboratory members for support and comments. We thank T. Oelschlaegel (Gurdon Institute, Cambridge University, UK) for polyclonal GFP–Flag–MRE11 U2OS cells, J. Forment (Gurdon Institute, Cambridge University, UK) for stable GFP–CtIP-WT U2OS cells, C. le Sage for helping to generate U2OS cells stably expressing GFP–CtIP variants (12KR, 6KR and 5KR), J. Travers (Gurdon Institute, Cambridge University, UK) for the pEGFP-C1/TO plasmid and for helping establish inducible GFP–RNF138 U2OS cells, the Y. Shiloh laboratory for the RPA2 mouse hybridoma, the Y. Shiloh (Department of Human Molecular Genetics and Biochemistry Sackler School of Medicine, Tel Aviv University, Israel) and M. Oren (The Weizmann Institute of Science, Israel) laboratories for HA–ubiquitin plasmid, the P. Cohen (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, UK) laboratory for UBE2T plasmid, L. Pellegrini and N. Rzechorzek (Department of Biochemistry, Cambridge University, UK) for providing baculovirus-purified CtIP, R. Carazo-Salas for access to the Opera, A. Riddell for flow-cytometry cell sorting support, M. Herzog and N. Smith for advice on Circos plots, the Gurdon Institute bioinformatics core facility, in particular C. Bradshaw and G. Allen, and the Gurdon Institute imaging facility, in particular A. Sossick, N. Lawrence and R. Butler. Research in the S.P.J. laboratory is funded by Cancer Research UK Program Grant C6/A11224, the European Research Council (DDREAM), the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010-259893 (DDResponse). Core infrastructure funding was provided by Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096. S.P.J. receives a salary from the University of Cambridge, supplemented by Cancer Research UK. C.K.S. was financially supported by a FEBS Return-to-Europe fellowship. P.B. is supported by the Emmy Noether Programme of the German Research Foundation (DFG, BE 5342/1-1).

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C.K.S./Y.G. and S.P.J. conceived the project. C.K.S./Y.G. performed the experiments with help from M.S.-C. and J.C. C.K.S./Y.G. analysed data. LC–MS/MS was by P.B. M.D., M.C. and S.J. generated reagents. C.K.S./Y.G. and S.P.J. wrote the manuscript. All authors made suggestions and commented on the manuscript.

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Correspondence to Yaron Galanty or Stephen P. Jackson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Table 1 Sequences of siRNA oligonucleotides used in this study.
Supplementary Table 2 HR and mutEJ repair efficiency measured by TLR assay.
Supplementary Table 3 Sequences of primers used in this study.
Supplementary Table 4 Antibodies used in this study.

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Schmidt, C., Galanty, Y., Sczaniecka-Clift, M. et al. Systematic E2 screening reveals a UBE2D–RNF138–CtIP axis promoting DNA repair. Nat Cell Biol 17, 1458–1470 (2015). https://doi.org/10.1038/ncb3260

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