RNF168 Ubiquitinates K13-15 on H2A/H2AX to Drive DNA Damage Signaling

• Published in: Cell 2012-09, Vol.150 (6), p.1182-1195
• Main Authors: Mattiroli, Francesca, Vissers, Joseph H.A., van Dijk, Willem J., Ikpa, Pauline, Citterio, Elisabetta, Vermeulen, Wim, Marteijn, Jurgen A., Sixma, Titia K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Histones - chemistry; Histones - metabolism; Humans; Lysine - metabolism; Models, Molecular; Molecular Sequence Data; Nucleosomes - chemistry; Nucleosomes - metabolism; Protein Structure, Tertiary; Scattering, Small Angle; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - metabolism; X-Ray Diffraction
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2012.08.005

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. [Display omitted] ► Recruitment order of RNF8/RNF168 is uncoupled from activity on nucleosomal H2A/H2AX ► A charged site on these E3s affects histone ubiquitination, but not chain formation ► RNF168 ubiquitinates K13-15 of H2A/H2AX at DSBs, distinct from K119 PRC1 site ► Target modification by RNF168 rather than Ub chain formation drives DSB signaling The RING domain of RNF168 recognizes H2A/H2AX at sites of double-strand DNA breaks. RNF168 ubiquitinates these histones at lysines 13–15 to enable the recruitment of downstream factors and drive DNA repair.