The chl1 mutation confers growth sensitivity in the presence of hydroxyurea without compromising the DNA replication checkpoint. A. Spot assay for HU sensitivity of 699 (wild-type) and 699Dchl1 (chl1). Growing cells were serially diluted and spotted on YEPD plates containing 0.1 M HU and no HU (YEPD). Plates were incubated at 30°C for 2 days (YEPD) or 4 days (YEPD+HU). B. The chl1 mutant shows moderate loss in cell viability upon HU treatment. 699 (wild-type), 699Dchl1 (chl1) and SL7 (rad53-21) cells were arrested by alpha-factor in G1 and released in fresh YEPD containing 0.2 M HU. Aliquots were removed for cell viabilities at the indicated time points. C. S-phase checkpoint is active in chl1 mutant cells. SL14 (CHL1) and SL14Dchl1 (chl1) were arrested in G1 phase and released in fresh YEPD medium containing 0.2 M HU at 30°C which was taken as 0 hour. Rad53p phosphorylation was detected by western blot analysis of proteins extracted from aliquots of cells removed after 0 and 2 hours of HU treatment, using antibodies directed against the Rad53 protein.