ABSTRACT
DNA double-strand breaks (DSBs) are deleterious lesions that lead to genetic mutations and cell death. Protein ubiquitination mediated by the E3 ubiquitin ligase RNF8 within the regions surrounding DSBs recruits DNA DSB response (DDR) factors and induces chromatin remodeling, which supports cell survival after DNA damage. Nevertheless, the impact of RNF8-mediated ubiquitination on DNA repair remains to be elucidated. Here, we report that depletion of the deubiquitinating enzyme OTUB2 enhances RNF8-mediated ubiquitination in an early phase of the DDR and promotes faster DSB repair but suppresses homologous recombination. The rapid ubiquitination results in accelerated accumulation of 53BP1 and RAP80 at DSBs, which in turn protects DSB ends from resection in OTUB2-depleted cells. Mechanistically, OTUB2 suppresses RNF8-mediated L3MBTL1 ubiquitination and Lys 63-linked ubiquitin chain formation in a deubiquitinating activity-dependent manner. Thus, OTUB2 fine-tunes the speed of DSB-induced ubiquitination so that the appropriate DNA repair pathway is chosen.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Thiolester Hydrolases/chemistry , Carrier Proteins/metabolism , Cell Death , Cell Line, Tumor , DNA-Binding Proteins/chemistry , Gene Library , Gene Silencing , HeLa Cells , Histone Chaperones , Histones/chemistry , Homologous Recombination , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lysine/chemistry , Mutation , Nuclear Proteins/metabolism , Plasmids/metabolism , RNA, Small Interfering/metabolism , Recombination, Genetic , Tumor Suppressor p53-Binding Protein 1 , Ubiquitin/chemistry , Ubiquitin-Protein LigasesABSTRACT
Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V-), rNiV(C-), and rNiV(W-), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V-) and rNiV(C-) were lower than the other recombinants. The rNiV(V-), rNiV(C-) and rNiV(W-) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V-) and rNiV(C-) but not the rNiV(W-) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo.
