Figure 4. The 9-1-1 clamp activates Mec1 in G2 cells via two distinct mechanisms.
(A) Log phase cells were arrested in G2 phase with nocodazole (20 μg/ml) for 3 hours at 30 °C, then treated with 4NQO for 20 min. A western analysis of Rad53 phosphorylation was carried out as described in Methods. The % of hyperphosphorylated Rad53 is shown beneath the lanes.
(B) as in (A), except that the entire experiment was carried out at 23 °C (dpb11-1 is temperature-sensitive for growth), nocodazole treatment was for 4 hours, and 4NQO treatment for 20 min.
(C) Ten-fold serial dilutions of wild type cells and the indicated DDC1 mutants were tested for sensitivity to UV (60 J/m2) or camptothecin (10 μg/ml). Plates were incubated at 30 °C for two days and photographed.