For a long time, it has been assumed that when cells are exposed to genotoxic agents, chromatin histones H2A and H2A.X are modified by a specific type of modification, i.e. K63-linked ubiquitination on the C-terminal K119 of H2As, by the ubiquitin ligase RNF168. This poses the issue of specificity in the system, due to the abundance of H2A ubiquitination (10-15% of H2As are ubiquitinated on K119). While interrogating this issue, we discovered that RNF168 targets non-canonical sites on histone H2A, namely K13 and K15, generating novel chromatin marks at the N-terminal tail of the protein (H2AK13ub and H2AK15ub, panel A). Moreover, we showed that an additional level of specificity is given by the peculiar type of ubiquitination required to trigger RNF168 activity, which is mediated by K27 linkage of ubiquitin (panel B). All together these findings account for the specificity of the signal triggered by DNA double strand breaks and involving the ubiquitin ligase RNF168.
(A) Mass spectrometry analysis on chromatin extracts of cells expressing RNF168 or the empty vector and identification of the novel K13/K15 ubiquitination site on histone H2A and H2A.X (Gatti, Pinato et al. 2012). (B) schematic representation of the K/R ubiquitin mutants and their effect on i) chromatin ubiquitination (see Western blotting with FLAG antibodies) and ii) formation of 53BP1 foci upon etoposide treatment (Gatti et al, 2015).