FIGURE 7.

DNA damage-induced CtIP phosphorylation is impaired in CtIP dimerization mutants. A, interaction of CtIP 1–897 or indicated mutants with DNA in vitro. Left panel, biotin-labeled DNA fragments (0.5 kb) were used in pull-down assays employing M-270 streptavidin Dynabeads and subjected to Western blotting using anti-CtIP antibody. Right panel, purified recombinants for this assay were resolved in SDS-PAGE (8% gel; Coomassie Blue staining). B, U2OS cells expressing HA-CtIP 1–897 or indicated mutants were treated with or without IR radiation (10 Gy) and allowed to recover for 1 h. After recovery, the cells were collected and lysed. Immunoblotting was performed using anti-HA antibody. C, purified GST-CtIP wild type or dimerization mutants were incubated with [γ-³²P]ATP in the presence or absence of ATM kinase and immunoprecipitated from 293T cells by anti-FLAG M2 beads for 30 min at 30 °C. The radiolabeled CtIP was visualized following SDS-PAGE.