RNF168-mediated H2A neddylation antagonizes ubiquitylation of H2A and regulates DNA damage repair.

NEDD8 is an important regulatory factor in many biological processes. However, the substrates for neddylation, and the relationship between the ubiquitin and NEDD8 pathways remain largely unknown. Here, we show that NEDD8 is covalently conjugated to histone 2A (H2A), and that neddylation of H2A antagonizes its ubiquitylation. NEDD8 suppresses ubiquitylation of H2A, and a decreased level of free NEDD8 promotes H2A ubiquitylation. Furthermore, we found that the E3 ligase RNF168 promotes both H2A ubiquitylation and neddylation. Interestingly, RNF168 is itself a substrate for NEDD8, and neddylation of RNF168 is necessary for its E3 ubiquitin activity. Inhibition of RNF168 neddylation impairs the interaction between RNF168 and its E2 enzyme Ubc13 (also known as UBE2N). Moreover, in response to DNA damage, the level of H2A neddylation decreased with an increase in the ubiquitylation of H2A, which facilitates DNA damage repair. During the later stages of damage repair, H2A neddylation increased gradually, whereas ubiquitylation decreased to basal levels. Mechanistically, NEDD8 negatively regulates the DNA damage repair process through suppression of the ubiquitylation of H2A and γH2AX, which further blocks the recruitment of the damage response protein BRCA1. Our findings elucidate the relationship of H2A ubiquitylation and neddylation, and suggest a novel modulatory approach to DNA damage repair through the neddylation pathway.