Extended Data Fig. 1: RNF168 prefers to ubiquitinate H2AK13/15 without polyubiquitin chain linkage preference.
a, In vitro ubiquitination assay to compare the activity of RNF1681–113 and RNF168FL to generate monoubiquitination (H2AUb) and dual-monoubiquitination (H2AUb2) on H2A K13/K15 NCP. b, In vitro ubiquitination assay shows that RNF1681–113 preferentially ubiquitinates H2AK13/15 rather than H2BK120. c, In vitro ubiquitination assay shows that RNF1681–113 together with UbcH5c can generate short ubiquitin chains on H2A K13R/K15 NCP. Note that there is only one lysine (H2A K15) on H2A K13/K15 NCP that can be conjugated with ubiquitin and bands of H2AUb3 were observed, suggesting that at least three ubiquitin were conjugated to H2A K15. The PRC1RING E3 ligase that site-specifically catalyzes nucleosomal H2A K118/K119 monoubiquitination was used as a negative control and it failed to generate significant H2A ubiquitination on H2A K13R/K15 NCP. d. In vitro ubiquitination assay to test the ubiquitin chain elongation activity of RNF1681–113 or RNF168FL on H2A K13R/K15Ub NCP. Note that in this nucleosome substrate, all the lysines on H2A were substituted by arginines so that ubiquitin can only be conjugated to the Ub motif at the H2A K15 site. It was observed that RNF1681–113 or RNF168FL generated H2AUb3, suggesting that no more than two Ub were conjugated to the Ub motif of the H2A K13R/K15Ub NCP and the ubiquitin chain elongation activity of RNF1681–113 or RNF168FL is weak. e,f, In vitro ubiquitination assays to investigate the ubiquitin chain linkage preference of RNF1681–113 on H2A K13R/K15 NCP (e) and H2A K13R/K15Ub NCP (f). These results suggest that RNF1681–113 generates short and mixed ubiquitin chains at H2A K15 sites with no linkage preference in vitro. For all the biochemical results, three independent experiments were performed with similar results.
