Fig 4.

Identification of acidic residues in H2AX and H2A that are required for ubiquitylation in cis. (A) fH2AX E92A exhibits reduced γH2AX formation upon DNA damage. Cells were transfected with the indicated vectors as in Fig. 3B and C. For DNA damage, transfected cells were treated with 60 μg/ml of phleomycin for 2 h and samples were collected at the indicated times post-phleomycin treatment. Samples were analyzed as in Fig. 3B and quantified as in Fig. 3D. Note that fH2AX E92A shows defective fH2AX-Ub and γH2AX formation. (B) Ubiquitylation of H2AX is reduced in H2AX E92A. Experiments were performed with the indicated constructs as in Fig. 1B. Cells were damaged as in panel A. (C) Mutations of specific acidic amino acids in H2AX affect its monoubiquitylation. The indicated fH2AX mutants were analyzed as in Fig. 2C. Cells were damaged as in panel A. (D) Mutation of specific acidic amino acids in H2A affects its monoubiquitylation. The indicated fH2A mutants were analyzed as in Fig. 2C. Cells were damaged as in panel A. (E) Acidic triad mutants of H2AX are sumoylated. Experiments were performed as in Fig. 1B by cotransfections with HA-SUMO1 and fH2AX mutants. Arrows identify H2AX and its modified forms as labeled. Note the shift of mobility of the H2AX sumoylation band in cells expressing HA-tagged SUMO1. (F) Molecular modeling of acidic triad motif mutants and Lys-119 ubiquitylation of H2A(X). Nucleosomes were modeled from the X-ray crystal structure (1.9 A) of a Xenopus nucleosome associated with human DNA (42). The structure was retrieved from a protein data bank (accession no. 1KX5). Modeling and coloring were performed in PyMol. The acidic triad motif refers to E61, D90, and E92 of H2A(X). Individual components of the model were colored to facilitate visualization as indicated in the legend.