Fig.1
Chl1p is required for G1/S after DNA damage by MMS. A G1-phase bud emergence kinetics of mutant and wild-type cells after MMS treatment. Wild-type (699) and mutant cell 699Dchl1 (chl1) were grown to exponential phase (~ 0.2 OD610nm) 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, each culture was divided into two. To one half 0.2% MMS was added and the other was maintained as a control. 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 and chl1 cells at different time intervals after release from G1 arrest and 0.2% MMS treatment simultaneously. The black filled symbols are given for cells treated with MMS, the grey filled symbols indicates the absence of MMS. Data shown are averages of values obtained from three independent experiments and the deviations from the mean are shown as error bars. B Growth of WT and mutant cells on YEPD plates. Wild-type (699) and mutant cell 699Dchl1 (chl1), SL3 (rad24) and SL3Dchl1 (rad24chl1) were streaked for single colony on YEPD plates and incubated at 30 °C for (i) 30 h, (ii) 34 h and (iii) 60 h respectively. We could observe an initial growth difference between the WT and the mutant, chl1 that goes of after 60 h of incubation C Budding of mutant and wild-type cells after MMS treatment. The bright fields of WT and chl1 from (A) at 40X resolution shows the budded cells in wild-type (699) and chl1 (699DChl1) mutant cultures after 1 and 2 h of release from MMS treatment. The budded cells are indicated with arrows. D Chl1 cells have fragmented DNA at G1 phase when treated with MMS. 699 (wild-type) and 699Dchl1 (chl1) cells were arrested at G1 by treating the log phase cells with alpha-factor for 90 min. To these G1 blocked cells, 0.2% MMS was added to create a substantial damage. Cells were collected at different time points of MMS exposure for DAPI staining. 0’ was collected just after adding 0.2% MMS to the cells with alpha-factor (G1-blocked) followed by 10’, 20’ and 30’ of exposure to 0.2% MMS in presence of alpha-factor (G1-blocked damaged cells). Representative fields of DAPI staining of cells treated for 0’ and 30’ with 0.2%MMS is given for WT and chl1 mutant cells. The corresponding bright field and merged images are also given along with the DAPI field. E chl1 cells are sensitive towards killing by genotoxic agent in G1/S-phase. 699 (wild-type), 699Dchl1 (chl1), SL3 (rad24), SL3Dchl1 (rad24chl1), 699Δsgs1 (sgs1), 699Δsgs1Dchl1 (sgs1chl1) and SL21 (sgs1rad24) cells were arrested by alpha-factor in G1. To these G1 blocked cells, 0.2% MMS was added to create a substantial damage at G1. Cells were collected at different time points of MMS exposure for viability assay. 0’ was collected just after adding 0.2% MMS to the cells with alpha-factor (G1-blocked) followed by 10’, 20’ and 30’ of exposure to 0.2% MMS in presence of alpha-factor (G1-blocked damaged cells). Aliquots removed for cell viabilities at the indicated time points were washed off of both MMS and alpha-factor, resuspended in water, counted and plated after dilution on YEPD plates. The plates were incubated at 30 °C for 2–3 days and the viable colonies were counted