Fig. 3
Chl1p plays a role in regulating the checkpoints at G1/S phase of the cell cycle. A G1/S-phase progression of mutant and wild-type cells in the presence of MMS. Wild-type (699) and mutant cell 699Dchl1 (chl1), SL3 (rad24) and SL3Dchl1 (rad24chl1) were all synchronized with alpha-factor at 30 °C and 0.2% MMS was added in presence of the G1 block. All the cultures were kept shaking at 30 °C. Aliquots were removed at various times for FACS analysis. The histogram plot at each time point are overlayed in the figure by using overlay software to understand the progression of the cells through cell cycle. The exponential cells were collected just before the addition of alpha-factor to the growing cells of 0.2 OD610nm. Arrows indicates G1 and G2 DNA contents. B chl1 cells are compromised in Rad53p phosphorylation in response to MMS treatment in G1/S-phase. Wild type, CHL1 (699) and 699Dchl1 (chl1) cells were arrested in G1 phase and exposed to 0.2% MMS at 30 °C. Rad53p phosphorylation was detected by western blot analysis of proteins extracted from aliquots of cells removed at indicated times, using antibodies directed against the Rad53 protein. C rad24chl1 cells are more compromised in Rad53p phosphorylation compared to chl1 cells in response to MMS treatment in G1/S-phase. SL3 (rad24) and SL3Dchl1 (rad24chl1) cells were arrested in G1 phase and exposed to 0.2% MMS at 30 °C along with the cells of B. Rad53p phosphorylation was detected by western blot analysis of proteins extracted from aliquots of cells removed at indicated times, using antibodies directed against the Rad53 protein. D Quantification of Rad53p expression in chl1 cells along with the double mutant rad24chl1 cells. The intensity of the phosphorylated bands of Rad53p in WT (CHL1), 699Dchl1 (chl1), SL3 (rad24) and SL3Dchl1 (rad24chl1) cells in western blots was quantified using Image J software. The values of the Rad53p phosphorylated band intensities taken together were normalized with corresponding intensities of beta-actin to normalize the protein loading at different time points. The 0 min is just after adding 0.2% MMS followed by 10, 20 and 30 min exposure to 0.2% MMS. The graph shows the average of data obtained from 3 repeated experiments