title: The deubiquitylating enzyme USP44 counteracts the DNA double-strand break response mediated by the RNF8 and RNF168 ubiquitin ligases.
abstract: Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.
authors.affiliation: Ubiquitin Signaling Group, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Anna
authors.initials: A
authors.lastname: Mosbech
authors.affiliation: Chromosome Stability and Dynamics Group, Department of Disease Biology, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Claudia
authors.initials: C
authors.lastname: Lukas
authors.affiliation: Ubiquitin Signaling Group, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address: simon.bekker-jensen@cpr.ku.dk.
authors.forename: Simon
authors.initials: S
authors.lastname: Bekker-Jensen
authors.affiliation: Ubiquitin Signaling Group, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address: niels.mailand@cpr.ku.dk.
authors.forename: Niels
authors.initials: N
authors.lastname: Mailand
mesh_terms: D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000072017:Deubiquitinating Enzymes; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C486521:RNF8 protein, human; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; D000072017:Deubiquitinating Enzymes; C538826:OTUB1 protein, human; C579223:USP29 protein, human; C579221:USP3 protein, human; D064570:Ubiquitin-Specific Proteases; D003546:Cysteine Endopeptidases
keywords: DNA Damage Response; DNA Repair; Deubiquitylation; Signaling; Ubiquitin