The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97–ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97–ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97–ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.
Bibliographical noteFunding Information:
The Ramadan Laboratory is supported by a Medical Research Council UK programme grant (MC_EX_MR/K022830/1) to K.R. J.O. was supported by a Swiss National Science Foundation grant (31003A_141197) to K.R., a Goodger and Schorstein Scholarship, University of Oxford, and a European Institute of Innovation and Technology short-term post-doctoral fellowship. S. K, J. F. and S. L were supported by the Cancer Research UK or MRC studentships. B. Vo., A. E. K. and I. D. C. T. are supported by Cancer Research UK Programme Grant C5255/A15935. C.G. was supported by the Danish Cancer Society (Grant No. R146-RP11394). R. F. is supported by Spanish Ministry of Innovation Science and Universities/EU-ERDF (SAF2016-80626-R), E. C. is supported by the Fundaci?n DISA, and E. H-C. is supported by an ACIISI pre-doctoral fellowship. We thank Ross Chapman for providing us with WT and 53BP1-deficient MCF7 cell lines. We are grateful to Pam Reynolds for support with the UV micro-irradiation system, to the Mechanical and Electronics Workshops (J Prentice and RG Newman) for assistance with construction of the system, to the Bioanalysis Core (Graham Brown and Ioland Vendrell) for the supports with microscopy and mass spectrometry and to the Radiation Biophysics Core for the help with X-ray radiation. We thank Benedikt Kessler for his constant support with mass spectrometry. We thank Jeremy Stark for providing us with GFP-reporter assays for HR and NHEJ.
© 2019 The Authors. Published under the terms of the CC BY 4.0 license
- Ataxin 3
- DNA double-strand break repair
- E3 ubiquitin ligase RNF8
- genome stability
- p97/VCP ATPase