H2A/H2AX lysine 13-15 ubiquitination by RNF168 is required for the DNA damage response.
Major finding: H2A/H2AX lysine 13-15 ubiquitination by RNF168 is required for the DNA damage response.
Concept: RNF8 recruits RNF168 to sites of damage but cannot monoubiquitinate nucleosomal H2A.
Impact: Establishing RNF168 as a priming ligase provides insight into the order of events after DNA damage.
Ubiquitination of histones H2A and H2AX at sites of DNA double-strand breaks (DSB) is required for the recruitment of downstream effectors of the DNA damage response (DDR), but the orchestration of early histone ubiquitination events remains incompletely understood. Ring finger protein 8 (RNF8) is the first E3 ubiquitin ligase recruited to damaged sites, where it is thought to initiate histone ubiquitination and recruit a second E3 ligase, RNF168, that extends the ubiquitin chains. To gain insight into the biochemical properties of RNF8- and RNF168-mediated histone ubiquitination, Mattiroli and colleagues reconstituted these reactions in vitro with purified human histones and oligonucleosomes. Surprisingly, they found that both E3 ligases could ubiquitinate free histone H2A, but only RNF168 was active toward nucleosomal H2A. Structural studies revealed that unlike RNF8, RNF168 has a positively charged amino acid residue C-terminal to its substrate recognition domain that is sufficient to confer the ability to ubiquitinate nucleosomal H2A. To determine the site where RNF168 ubiquitinates H2A, the authors performed in vitro ubiquitination reactions with RNF168 and oligonucleosomes containing mutated H2A variants. RNF168-dependent monoubiquitination was observed on lysines 13 or 15, not the previously identified lysine 119 site targeted by other E3 ligases. Induction of H2A lysine 13-15 ubiquitination was also observed in mammalian cells after DNA damage and was required for recruitment of DDR proteins. In cells, H2A lysine 13-15 ubiquitination was dependent on both RNF168 and RNF8, and further in vitro ubiquitination experiments showed that RNF8 extended ubiquitin chains on H2A following monoubiquitination by RNF168. This study thus identifies an additional histone modification involved in DSB signaling and suggests that a 2-step process occurs following DNA damage whereby RNF168 monoubiquitination primes histone H2A for RNF8-dependent ubiquitin chain extension.