E4 ligase–specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis

Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome. In the absence of UFD-2, RAD-51 foci persist, and DNA damage-induced apoptosis is prevented. In contrast, UFD-2 foci are retained until recombination intermediates are removed by the Holliday-junction-processing enzymes GEN-1, MUS-81 or XPF-1. Formation of UFD-2 foci also requires proapoptotic CEP-1 (p53) signaling. Our findings establish a central role of UFD-2 in the coordination between the DNA-repair process and the apoptotic response

The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97-UFD1-NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability

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

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. </p

ESCRT-III controls nuclear envelope reformation

During telophase, the nuclear envelope (NE) reforms around daughter nuclei to ensure proper segregation of nuclear and cytoplasmic contents(1-4). NE reformation requires the coating of chromatin by membrane derived from the Endoplasmic Reticulum and a subsequent annular fusion step to ensure the formed envelope is sealed(1,2,4,5). How annular fusion is accomplished is unknown, but it is thought to involve the p97 AAA-ATPase complex and bears a topological equivalence to the membrane fusion event that occurs during the abscission phase of cytokinesis(1,6). We find here that the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery localises to sites of annular fusion in the forming NE and is necessary for proper post-mitotic nucleo-cytoplasmic compartmentalisation. The ESCRT-III component Charged Multivesicular Body Protein (CHMP) 2A is directed to the forming NE through binding to CHMP4B and provides an activity essential for NE reformation. Localisation also requires the p97 complex member Ubiquitin Fusion and Degradation 1 (UFD1). Our results describe a novel role for the ESCRT-machinery in cell division and demonstrate a conservation of the machineries involved in topologically equivalent mitotic membrane remodeling events