title: Ubiquitin-H2AX fusions render 53BP1 recruitment to DNA damage sites independent of RNF8 or RNF168.
abstract: The mammalian E3 ubiquitin ligases RNF8 and RNF168 facilitate recruitment of the DNA damage response protein 53BP1 to sites of DNA double-strand breaks (DSBs). The mechanism involves recruitment of RNF8, followed by recruitment of RNF168, which ubiquitinates histones H2A/H2AX on K15. 53BP1 then binds to nucleosomes at sites of DNA DSBs by recognizing, in addition to methyl marks, histone H2A/H2AX ubiquitinated on K15. We report here that expressing H2AX fusion proteins with N-terminal bulky moieties can rescue 53BP1 recruitment to sites of DNA DSBs in cells lacking RNF8 or RNF168 or in cells treated with proteasome inhibitors, in which histone ubiquitination at sites of DNA DSBs is compromised. The rescue required S139 at the C-terminus of the H2AX fusion protein and was occasionally accompanied by partial rescue of ubiquitination at sites of DNA DSBs. We conclude that recruitment of 53BP1 to sites of DNA DSBs is possible in the absence of RNF8 or RNF168, but still dependent on chromatin ubiquitination.
authors.affiliation: a Department of Molecular Biology; University of Geneva ; Geneva , Switzerland.
authors.forename: Maciej K
authors.initials: MK
authors.lastname: Kocyłowski
authors.forename: Alix J
authors.initials: AJ
authors.lastname: Rey
authors.forename: Grant S
authors.initials: GS
authors.lastname: Stewart
authors.forename: Thanos D
authors.initials: TD
authors.lastname: Halazonetis
mesh_terms: D002460:Cell Line; D004249:DNA Damage / Q000235:genetics* / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D055786:Gene Knockout Techniques; D006657:Histones / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012394:Rosaniline Dyes; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C486521:RNF8 protein, human; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D025801:Ubiquitin; C048139:Coomassie blue; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: 53BP1; RNF168; RNF8; chromatin; ubiquitin