The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8.

Abhay Narayan Singh,Shudong Li,John Fielden,Claire Guérillon,Anne E Kiltie,Bruno Vaz,Borivoj Vojnovic,Susan Kilgas,Ignacio Torrecilla,Elisa Cabrera,Iain Dc Tullis,Esperanza Hernandez‐Carralero,Paul R Barber,Jason Parsons,Niels Mailand,Mayura Meerang,Raimundo Freire,Kristijan Ramadan,Judith Oehler

doi:10.15252/embj.2019102361

Abstract

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.

Highlights

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair
Under genotoxic conditions, when RNF8 is rapidly recruited to sites of DNA lesions and orchestrates the DNA damage response, the p97–Ataxin 3 (ATX3) complex facilitates RNF8 chromatin extraction to balance double-strand breaks (DSBs) repair pathway choice and improve cell survival to ionising radiation (IR)
The cells were biochemically fractionated into total soluble and chromatin pools, and Flag-RNF8 was subsequently immunopurified under denaturing conditions from both fractions to analyse its ubiquitination status (Fig EV1D)

Summary

Introduction

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. 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

Methods

Results

Conclusion