Figure 3: Targeting H2A requires an intact and accessible acidic patch.

(a) Position of the acidic patch within the nucleosome. NCP structure (1KX5 (ref. 55)) where H2B is shown in orange, H2A in yellow–orange, H3 in light pink and H4 in violet. K118–119 on H2A are highlighted in blue, K13–15 are in green and the glutamate residues of the acidic patch are shown in red. Distance between K13–15 and the acidic patch are indicated on the left. Electrostatic potential (red is negative, blue is positive) of the histone core in the NCP highlights the acidic patch on the surface of the H2A/H2B dimer. (b) Mutations in the acidic patch on H2A or H2B impair H2A ubiquitination in H2A/H2B dimers. Time course experiment (30–60–90 min) following activity of RNF168 RING domain towards H2A in the different H2A/H2B mutant dimers. (c) Acidic patch mutations prevent ubiquitination of NCPs. Time course experiment (30–60–90 min) to investigate the activity of RNF168 RING domains towards H2A in nucleosome WT or EA (H2A E61, 64, 91 Å and H2B E102, 110 Å). (d) Binding of a LANA peptide, but not a mutant LRS variant, to the nucleosome competes with RNF168-dependent ubiquitination of NCPs. Ubiquitination assay with 3–10–30–100 μM peptide. Note that E91 on H2A is part of the epitope recognized by the anti-H2A antibodies; therefore, there is reduced signal on western blots when the E91A mutation is introduced.