FIGURE 2.

TORC2-Gad8 is required for deactivation of the DNA damage checkpoint but not for its activation. A and B, Tor1 or Gad8 are dispensable for maintaining cellular viability in response to short exposure to MMS (A) or CPT (B). Cells were grown to log phase and shifted to 0.03% MMS or to 30 μm CPT for 6 h. Samples were taken every hour to determine cell viability by assessing plating efficiency on rich medium. C, logarithmic growing cells were transferred to rich medium containing either 30 μm CPT or 0.03% MMS. Samples of the indicated strains, before and after a 6-h exposure to the drugs, were stained with Hoechst and Calcofluor to visualize nuclei and septa, respectively. D, Tor1 is dispensable for Chk1 phosphorylation in response to CPT. Wild type or Δtor1 cells containing an HA-tagged Chk1 were grown to log phase and treated with 30 μm CPT. Protein extract from each of the indicated time points was analyzed by Western blot. The resulting membranes were probed with anti-HA to visualize Chk1 and with anti-Cdc2 as a loading control. E, the dephosphorylation of Chk1 is delayed in a Δtor1 mutant. Cells were arrested for 3 h in 30 μm CPT, washed thoroughly, and resuspended in fresh medium. Protein samples from the indicated time points were analyzed by Western blot as described above.