Fig. 1. Rad4 is required for activation of Cds1 and Chk1 kinases. (A) Wild-type and rad4-116 cells were synchronized in G2, released into pre-warmed media and grown in the absence (-) or presence (+) of 10 mM HU at the temperatures indicated. Whole cell extracts from S-phase cells were prepared and assayed for Cds1p kinase activity. The data show that Cds1p kinase activity is severely reduced or absent in rad4-116 cells at 32°C and 36°C, respectively, indicating that Rad4p is required for the activation of Cds1p kinase. (B) Wild-type and rad4-116 cells carrying an integrated HA-chk1⁺ allele were grown overnight to mid-log and then released into pre-warmed media and grown in the absence (-) or presence (+) of 20 µg/ml bleomycin sulphate or irradiated with 150 J/m² UV. Cells were also exposed to either a fixed dose of -irradiation 125 Gy (C) or doses in the range 0-125 Gy (D) at the temperatures indicated. Total protein was prepared from all cells and Chk1 phosphorylation analysed by immunoblot using anti-HA antibody (B-D).