title: RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling.
abstract: Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.
authors.affiliation: Division of Biochemistry and Center for Biomedical Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors.forename: Francesca
authors.initials: F
authors.lastname: Mattiroli
authors.forename: Joseph H A
authors.initials: JH
authors.lastname: Vissers
authors.forename: Willem J
authors.initials: WJ
authors.lastname: van Dijk
authors.forename: Pauline
authors.initials: P
authors.lastname: Ikpa
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Citterio
authors.forename: Wim
authors.initials: W
authors.lastname: Vermeulen
authors.forename: Jurgen A
authors.initials: JA
authors.lastname: Marteijn
authors.forename: Titia K
authors.initials: TK
authors.lastname: Sixma
mesh_terms: D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009707:Nucleosomes; C486521:RNF8 protein, human; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases; D008239:Lysine