Viability assays of Rad53 phosphomimic mutants upon MMS and HU treatment.

Fig 5A. Domain organizations of C. albicans Rad53 and S. cerevisiae Rad53. Arrowheads mark [S/T]Q amino acid mutant site. Amino acids at domain boundaries are indicated by numbers. Schematic description of the strategy for integrating RAD53 wild-type and mutant alleles at the RAD53 chromosomal locus (for details, see Materials and Methods.). Fig 5B. Cells of wild-type (S5314 or BWP17), rad53Δ (WY3), the rescued RAD53 (HT6) and the various strains expressing mutant alleles of RAD53 (HT7–18 refer to Table 1) were 10-fold serially diluted, spotted onto YPD plates containing different concentrations of HU or MMS, and incubated at 30°C for 24 h. Fig 5C. Approximately equal numbers of cells of wild type (S5314 or BWP17), pph3Δ (SJL3), rad53Δ (WY3), the rescued RAD53 (HT6) and the various strains expressing mutant alleles of RAD53 (HT13–16 rad53-S461A, rad53-S461D, rad53-S545A, rad53-S545D) were treated with 20 mM HU or 0.02 mM for 2 h in liquid culture and then spread onto YPD plates for incubation at 30°C for 2 d. Percentage of viability was expressed as CFU of the untreated mutants compared to untreated wild-type cells, and CFU of HU-treated or MMS-treated mutants was compared to their untreated counterpart. All data show the average of three independent experiments with error bars. Fig 5D. Cells of wild type (S5314 or BWP17), pph3Δ (SJL3), rad53Δ (WY3), the rescued RAD53 (HT6) and the various strains expressing mutant alleles of RAD53 (HT13–16 rad53-S461A, rad53-S461D, rad53-S545A, rad53-S545D) were grown in liquid YPD medium supplemented with 0.02% MMS or 20 mM HU at 30°C for 4 h. Cells were collected for microscopic examination. 0.02% MMS or 20 mM HU treated cells washed with fresh YPD and recovered into MMS-free and HU-free YPD at 30°C for 8 h. Cells were collected for microscopic examination. (Bar = 5 µm)