Figure 6.—

Western blots of Rad53p in Ofm mutants. Portions of Western blots probed to detect Rad53p are shown. The hyperphosphorylation of Rad53p, indicating activation of the checkpoint, is detected as a decrease in electrophoretic mobility. (Left, 4-NQO) Log-phase cells were arrested in G₁ with 20 μg/ml α-factor; after arrest, the UV mimetic, 4-nitroquinoline 1-oxide, was added to a final concentration of 2 μg/ml, and samples were taken at 0, 15, 30, 45, and 60 min; FACS analysis confirmed that cells remain blocked in G₁. Note that only the ofm14 mutant failed to hyperphosphorylate Rad53p under these conditions, indicating that the signal transduction cascade is blocked prior to Rad53p activation. (Right, HU) log-phase cells were first synchronized in G₁ with α-factor and then released from the G₁ block into medium containing 0.2 m hydroxyurea, and samples were taken at 0, 30, 60, 120, and 180 min, except for the ofm2 time course in which samples were taken at 20, 40, 60, 90, and 120 min. Note that Rad53p was hyperphosphorylated by all the strains, indicating that the replication stress signal transduction cascade is intact.