Fig. 4

Chl1p acts independently of the DNA damage checkpoint pathway. A G1-phase bud emergence kinetics of Chl1 mutant cells are additive to rad24 after MMS treatment. Wild-type (699) and mutant cells 699Dchl1 (chl1), SL3 (rad24), SL3Dchl1 (rad24chl1) were grown to exponential phase and arrested with 5 μg/ml α-factor for 90 min (G1 arrest) as described in materials and methods. After 80 min of α-factor treatment at 30 °C, 0.2% MMS was added. Cells were kept shaking for a further 10 min. After treatment, MMS was inactivated by the addition of one volume of 10% sodium thiosulfate solution, cells were spun down and the pellet was washed quickly with YEPD medium at RT. The cells were released in a fresh YEPD medium at 30 °C and aliquots were removed at regular times for scoring the percentage of budded cells. The graph represents the percentage of bud emergence in WT, chl1, rad24 and rad24chl1 cells at different time intervals after release from G1 arrest and 0.2% MMS treatment simultaneously. Data shown are the average of values obtained from three independent experiments. B G1-phase bud emergence kinetics of cells in absence of MMS treatment. Wild-type (699) and mutant cells 699Dchl1 (chl1), SL3 (rad24), SL3Dchl1 (rad24chl1) were simultaneously grown with the A cells to exponential phase and arrested with 5 μg/ml α-factor for 90 min (G1 arrest). The cells were released in a fresh YEPD medium without any MMS treatment at 30 °C and aliquots were removed at regular times for scoring the percentage of budded cells. C G1-phase bud emergence kinetics of Chl1 mutant cells is in line with rad9 after MMS treatment. Wild-type (699) and mutant cells 699Dchl1 (chl1), SL19 (rad9), SL19Dchl1 (rad9chl1) were grown to exponential phase and follow through same experimental procedures as done in A. The graph represents the percentage of bud emergence in WT, chl1, rad9 and rad9chl1 cells at different time intervals after release from G1 arrest and 0.2% MMS treatment simultaneously. Data shown are average of values obtained from three independent experiments. D G1-phase bud emergence kinetics of cells in absence of MMS treatment. Wild-type (699) and mutant cells 699Dchl1 (chl1), SL19 (rad9), SL19Dchl1 (rad9chl1) were simultaneously grown with the C cells to exponential phase and arrested with 5 μg/ml α-factor for 90 min (G1 arrest). The cells were released in a fresh YEPD medium without any MMS treatment at 30 °C and aliquots were removed at regular times for scoring the percentage of budded cells. E Additive and synergistic budding of mutant and wild-type cells after MMS treatment. The bright fields of WT and mutant cells from (A, B) at 40X resolution shows the budded cells in wild-type (699) and the mutant cells 699Dchl1 (chl1), SL3 (rad24), SL3Dchl1 (rad24chl1), SL19 (rad9), SL19Dchl1 (rad9 chl1) mutant cultures after 2 h release from MMS treatment. The budded cells are indicated with arrows