The nucleosome acidic patch plays a critical role in RNF168-dependent ubiquitination of histone H2A

During DNA damage response, the RING E3 ligase RNF168 ubiquitinates nucleosomal H2A at K13–15. Here we show that the ubiquitination reaction is regulated by its substrate. We define a region on the RING domain important for target recognition and identify the H2A/H2B dimer as the minimal substrate to confer lysine specificity to the RNF168 reaction. Importantly, we find an active role for the substrate in the reaction. H2A/H2B dimers and nucleosomes enhance the E3-mediated discharge of ubiquitin from the E2 and redirect the reaction towards the relevant target, in a process that depends on an intact acidic patch. This active contribution of a region distal from the target lysine provides regulation of the specific K13–15 ubiquitination reaction during the complex signalling process at DNA damage sites.