Figure 2.

DNA-damage-dependent phosphorylation of Pds1p-HA is Mec1p- and Rad9p-dependent. (A) Wild-type (OCF1522), mec1–1 (OCF1523), and mec1–1 cells carrying a centromere-based plasmid (pRS313, indicated as vector) or a pRS313 derivative carrying a copy of the wild-type MEC1 gene (pOC75, indicated as MEC1) were grown at 30°C and were arrested in G₁ phase with the α mating pheromone. When more than 85% of the cells appeared arrested morphologically (shmooed), the cultures were either left untreated (−) or exposed to γ-radiation (+, 4 krad). After the irradiation, the cultures were released into medium containing Pronase and nocodazole. Samples for protein extracts were taken 2 h after the release from G₁ phase and analyzed by Western blot analysis. At the time when the sample were taken, approximately 90% of the cells in all cultures had a mitotic arrest phenotype (large budded cell with a single nucleus) as determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. (B) Wild-type cells (OCF1522), rad9 cells (OCF1544), and rad9 cells carrying a centromere-based plasmid (pRS314, indicated as vector) or a pRS314-derived plasmid containing a copy of the RAD9 wild-type gene (pOC81, indicated as RAD9) were treated as described in A.