Mutation of the acidic patch impairs human H2AX and H2A ubiquitination.

<p>(<b>A</b>) Schematic of all H2AX lysines (K) and mutant derivatives. allR represents an all lysine (K) to arginine (R) version of H2AX. Additional site-specific reversions from arginine to lysine within the allR H2AX derivate are indicated. (<b>B</b>) H2AX-allR and acidic patch mutation E92A reduces H2AXub. WT or E92A H2AX/H2A constructs were transfected into HEK293T cells and analyzed by western blotting with the indicated antibodies. Arrows indicate ub forms. (SFB = S-tag, Flag epitope tag, and streptavidin-binding peptide tag; e = endogenous). Molecular weights (kDa) are indicated on the left of each panel. HEK293T cells were used for all cellular assays. (<b>C</b>) H2AX-K13/15 dependent ubiquitination requires the acidic patch. H2AX and derivatives were expressed in HEK293T cells (−) or (+) ionizing radiation (IR, 20 Gy). Samples were analyzed as in A 6 h post-IR treatment. (<b>D</b>) H2AX-K13/15 and K118/119-dependent ubiquitination requires the acidic patch. Cells were co-transfected with H2AX and derivatives along with Myc-RNF168 and analyzed as in C. (<b>E</b>) Phospho-competent H2AX S139 is not required <i>in cis</i> for H2AX K13/15ub. Cells were analyzed as in C. tub = tubulin loading control.