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Pph3 dephosphorylation of Rad53 is required for cell recovery from MMS-induced DNA damage in Candida albicans.

Wang H, Gao J, Li W, Wong AH, Hu K, Chen K, Wang Y, Sang J - PLoS ONE (2012)

Bottom Line: The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role.Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated.We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, People's Republic of China.

ABSTRACT
The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role. Here we report that the Pph3/Psy2 phosphatase complex, known to be involved in Rad53 dephosphorylation, is required for cellular responses to the DNA-damaging agent methyl methanesulfonate (MMS) but not the DNA replication inhibitor hydroxyurea (HU) in C. albicans. Deletion of either PPH3 or PSY2 resulted in enhanced filamentous growth during MMS treatment and continuous filamentous growth even after MMS removal. Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated. We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS. Therefore, our studies have identified a novel molecular mechanism mediating DNA damage response to MMS in C. albicans.

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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)
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pone-0037246-g005: 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)

Mentions: To identify potential phosphorylation sites on Rad53 that may be responsible for MMS sensitivity, we performed phosphomimetic mutagenesis on previously reported phosphorylation sites (Fig. 5A). Results showed that the Rad53 phosphomimetic mutants of S461D and S545D (corresponding to 489 and 560 in S. cerevisiae, GeneID 855950) exhibited higher sensitivity to MMS but not HU than the wild-type strain (Fig. 5B). Viability of these mutants also dropped dramatically after MMS treatment, but they were able to recover from HU treatment (Fig. 5C, Table 2). Moreover, the two mutants remained in pseudohyphal form 10 h after MMS withdrawal, but could fully return to the yeast growth after HU treatment (Fig. 5D, Table 2).


Pph3 dephosphorylation of Rad53 is required for cell recovery from MMS-induced DNA damage in Candida albicans.

Wang H, Gao J, Li W, Wong AH, Hu K, Chen K, Wang Y, Sang J - PLoS ONE (2012)

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)
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pone-0037246-g005: 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)
Mentions: To identify potential phosphorylation sites on Rad53 that may be responsible for MMS sensitivity, we performed phosphomimetic mutagenesis on previously reported phosphorylation sites (Fig. 5A). Results showed that the Rad53 phosphomimetic mutants of S461D and S545D (corresponding to 489 and 560 in S. cerevisiae, GeneID 855950) exhibited higher sensitivity to MMS but not HU than the wild-type strain (Fig. 5B). Viability of these mutants also dropped dramatically after MMS treatment, but they were able to recover from HU treatment (Fig. 5C, Table 2). Moreover, the two mutants remained in pseudohyphal form 10 h after MMS withdrawal, but could fully return to the yeast growth after HU treatment (Fig. 5D, Table 2).

Bottom Line: The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role.Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated.We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, People's Republic of China.

ABSTRACT
The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role. Here we report that the Pph3/Psy2 phosphatase complex, known to be involved in Rad53 dephosphorylation, is required for cellular responses to the DNA-damaging agent methyl methanesulfonate (MMS) but not the DNA replication inhibitor hydroxyurea (HU) in C. albicans. Deletion of either PPH3 or PSY2 resulted in enhanced filamentous growth during MMS treatment and continuous filamentous growth even after MMS removal. Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated. We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS. Therefore, our studies have identified a novel molecular mechanism mediating DNA damage response to MMS in C. albicans.

Show MeSH
Related in: MedlinePlus