Figure 1.
pph3Δ and psy2Δ cells exhibit pseudohyphal growth and cell cycle arrest when treated with MMS or HU.
Fig 1A. Wild-type (SC5314 or BWP17), pph3Δ (SJL3) and psy2Δ (SJL6) cells were grown in liquid YPD medium supplemented with 0.02% MMS or 20 mM HU at 30°C for 6 h, washed with fresh YPD and resuspended into fresh YPD for further growth at 30°C for 8 h. Bud length was measured using ImageJ (http://rsbweb.nih.gov/ij/index.html). Each data point represents the average of 30 cells measured in 3 independent experiments. Fig 1B. The same cells as used in (A) were treated with 0.02% MMS or 20 mM HU. Cells were harvested at indicated time intervals for flow cytometry analysis. Fig 1C. The same cells as used in (A) were treated with 0.02% MMS or 20 mM HU at 30°C for 6 h and recovered in fresh YPD as described in (A). Cells were harvested at indicated time intervals for flow cytometry analysis.
Table 1.
C. albicans strains used in this study.
Figure 2.
Rad53 undergoes hyperphosphorylation in response to HU and MMS.
Fig 2A. Rad53 hyperphosphorylation in HU-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 20 mM HU for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control. Fig 2B. Rad53 hyperphosphorylation in MMS-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 0.02% MMS for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control.
Figure 3.
Detection of Rad53 downstream signalling by RT-PCR, Northern blot and qPCR in pph3Δ and psy2Δ cells.
Fig 3A. Wild-type (S5314 or BWP17), pph3Δ (SJL3) and psy2Δ (SJL6) cells were incubated at 30°C in YPD containing 0.02% MMS and then recovered with fresh YPD over the indicated time period. RNA extracted from harvested cells at indicated time points was used for RT-PCR analysis. PCR amplifications in absence of retrotranscriptase for each sample was used as negative controls in RT-PCR. GAPDH was used as loading control. Fig 3B. Wild-type (S5314 or BWP17), pph3Δ (SJL3) and psy2Δ (SJL6) cells were incubated at 30°C in YPD containing 0.02% MMS for 6 h and then recovered with fresh YPD for 6 h. RNA was extracted and subject to Northern blot analysis. GAPDH was used as control and rRNA was shown to indicate RNA integrity. Fig 3C. Wild-type (S5314 or BWP17), pph3Δ (SJL3) and psy2Δ (SJL6) cells were incubated at 30°C in YPD containing 0.02% MMS for 6 h and then recovered with fresh YPD over the indicated time period. RNA was extracted and reverse transcribed into cDNA at the indicated time points for qPCR analysis. All data represent the mean of 3 independent experiments.
Figure 4.
Rfa2 was downregulated in pph3Δ and pph3Δ ptc2Δ mutant upon MMS but not HU treatment.
Fig 4A. Wild-type (HT1), pph3Δ (HT2), psy2Δ (HT3), ptc2Δ (HT4) and pph3Δ ptc2Δ (HT5) cells 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 4B. Approximately equal numbers of yeast cells were spread onto YPD plates containing different concentrations of HU and MMS for incubation at 30°C for 2 d. Percentage of viability was expressed as colony-forming units (CFU) of HU- or MMS- treated mutants compared to untreated wild-type control. Fig 4C. Wild-type (HT1), pph3Δ (HT2), psy2Δ (HT3), ptc2Δ (HT4) and pph3Δ ptc2Δ (HT5) cells expressing C-terminally Myc-tagged Rfa2 were incubated at 30°C in YPD containing 20 mM HU and recovered with fresh YPD over the indicated time period. Untreated cells was used as control. Total protein was extracted from harvested cells at the indicated time points and subject to immunoblot analysis with anti-Myc antibody. Cdc28 was probed with anti-PSTAIRE antibody as loading control. Fig 4D. Wild-type (HT1), pph3Δ (HT2), psy2Δ (HT3), ptc2Δ (HT4) and pph3Δ ptc2Δ (HT5) cells expressing C-terminally Myc-tagged Rfa2 were incubated at 30°C in YPD containing 0.02% MMS and recovered with fresh YPD over the indicated time period. Untreated cells were used as control. Total protein was extracted from harvested cells at the indicated time points and subject to immunoblot analysis with anti-Myc antibody. Cdc28 was probed with anti-PSTAIRE antibody as loading control.
Figure 5.
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)
Table 2.
Effects of the Rad53 S/T mutations on DNA checkpoint-mediated function.
Figure 6.
Detection of Rad53 downstream signaling by qPCR and Western blotting in pph3Δ cells and cells expressing various mutant alleles of RAD53 upon MMS treatment.
Fig 6A. Wild-type (SJL9), pph3Δ (SJL7), and the various strains expressing C-terminally Myc-tagged mutant alleles of RAD53 (HT13.1–16.1 rad53-S461A, rad53-S461D, rad53-S545A, rad53-S545D cells were incubated at 30°C in YPD containing 0.02% MMS for 4 h, then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control. Fig 6B. 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 were incubated at 30°C in YPD containing 20 mM HU and 0.02% MMS for 4 h, and then recovered with fresh YPD for 6 h. RNA was extracted and reverse transcibed into cDNA for qPCR analysis. All data represent the mean of 3 independent experiments. (* P value<0.01; ** P value<0.05). Fig 6C. Cells of wild type (HT1), pph3Δ (HT2) and the various RAD53 mutants expressing C-terminally Myc-tagged Rfa2 (HT21–24 rad53-S461A Rfa2-myc, rad53-S461D Rfa2-myc, rad53-S545A Rfa2-myc and rad53-S545D Rfa2-myc) were incubated at 30°C in YPD containing 0.02% MMS for 4 h and recovered with fresh YPD over the indicated time period. Untreated cells were used as control. Total protein was extracted from harvested cells at the indicated time points and subject to immunoblot analysis with anti-Myc antibody. Cdc28 was probed with anti-PSTAIRE antibody as loading control.