Figure 6.

Cds1 and Chk1 are both activated in polαts mutants at 36°C. (A) Phenotype of double mutant DB242 (polαts13 chk1Δ) at the restrictive temperature. Midlog phase double mutant DB242 (polαts13 chk1Δ) grown at 25°C was shifted to 36°C for 6 h. Cells were stained with DAPI and calcofluor. (B) Activation of p56^chk1:ep in DB232. Thirty micrograms of protein from cell lysates of DB232 carrying polαts13 and epitope-tagged chk1⁺, DBts13 (polαts13), and NW222 containing epitope-tagged chk1⁺ were fractionated on 8% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and probed with the 12CA5 antibody, and p56^chk1:ep was detected using the ECL system (Amersham, Arlington Heights, IL). The unphosphorylated p56^chk1:ep is marked by arrow a, and the phosphorylated p56^chk1:ep is marked by arrow b. Lanes 1 and 3, Strains NW222 and DB232 (polαts13 p56^chk1:ep) treated with MMS; lane 2, DBts13 (polαts13) containing no epitope-tagged chk1⁺ as a control; lanes 4–7, lysates from DB232 (polαts13 p56^chk1:ep) after 0, 2, 4, and 6 h at 36°C; lane 8, phosphatase-treated lysates from DB232 (polαts13 p56^chk1:ep) grown at 36°C for 6 h. (C) Activation of Cds1 kinase activity at 36°C. Cells were grown to midlog phase at 25°C and then shifted to 36°C for 3 h. Cds1 kinase activity was measured in wild-type cells, (wt), polαts11, and polαts13 as described in MATERIALS AND METHODS.