Fig 3.

Comprehensive screen for residues in H2AX that affect its ubiquitylation, sumoylation, and/or S139 phosphorylation. (A) Schematic outline of the H2AX screen. The amino acid sequence of H2AX is shown with individually mutated residues highlighted in one of 4 colors to indicate its category (see the text and legend for details). The diagram on the right graphically represents the protocol for the screen. (B) Complete H2AX screen in untreated and DNA damage conditions. The indicated constructs containing a single-amino-acid substitution to alanine in fH2AX, along with either vector or a wild-type fH2AX control, were transfected into U2OS cells. Cells were either untreated or treated with 60 μg/ml of phleomycin (Phleo) for 2 h. Whole-cell extracts were analyzed by Western blotting with Flag- and γH2AX-specific antibodies. Phospho S139, SUMO, and ubiquitylated (Ub) and unmodified (full-length) species of fH2AX are indicated. (C) Confirmation of positive hits from the H2AX screen. Mutants from panel B that affected any of the 3 PTMs analyzed in the screen compared to the wild type were reanalyzed as in panel B. (D) Quantification of γH2AX levels. The signal from full-length fH2AX from panel C was quantified. The signal from γH2AX was quantified, normalized to full-length levels, and plotted. Samples showing a greater than 25% difference in DNA damage-induced γH2AX compared to wild-type fH2AX were considered positive and are shown in red. (E) Quantification of H2AX ubiquitin levels. H2AX-Ub signals from panel C were quantified, normalized to full length, and compared to wild-type H2AX-Ub levels. Mutants whose Ub levels were greater than 25% different from wild-type H2AX-Ub levels were considered positive and are labeled in red. (F) Results of the H2AX screen. Mutants reproducibly affecting either of three PTMs of H2AX in both experiments from panels B and C are listed (see the text for details).