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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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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.
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pone-0037246-g006: 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.

Mentions: To gain evidence that phosphorylation at the above residues on Rad53 is biologically relevant, we first confirmed that the S to D mutants resulted in sustained Rad53 hyperphosphorylation after MMS treatment and recovery (Fig. 6A) but not HU (Fig. S4). Next, we used qPCR to examine the expression of Rad53 downstream genes after MMS treatment and recovery. qPCR results showed that RFA2 as well as the cyclin genes of CCN1 and PCL2 were downregulated in both Rad53-S461D and Rad53-S545D mutants after MMS treatment and during the recovery, while there was no obvious difference between the Rad53-S461A and Rad53-S545A mutants and the wild type (Fig. 6B). Western blotting analysis revealed that Rfa2 levels were not recovered in the Rad53-S461D and Rad53-S545D mutants, similar to results obtained in the pph3Δ mutant (Fig. 6C). Hence, our results strongly suggest that failure of dephosphorylation at S461 and S545 on Rad53 may be responsible for the MMS-induced pseudohyphal growth and sensitivity in pph3Δ and psy2Δ mutants.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351423&req=5

pone-0037246-g006: 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.
Mentions: To gain evidence that phosphorylation at the above residues on Rad53 is biologically relevant, we first confirmed that the S to D mutants resulted in sustained Rad53 hyperphosphorylation after MMS treatment and recovery (Fig. 6A) but not HU (Fig. S4). Next, we used qPCR to examine the expression of Rad53 downstream genes after MMS treatment and recovery. qPCR results showed that RFA2 as well as the cyclin genes of CCN1 and PCL2 were downregulated in both Rad53-S461D and Rad53-S545D mutants after MMS treatment and during the recovery, while there was no obvious difference between the Rad53-S461A and Rad53-S545A mutants and the wild type (Fig. 6B). Western blotting analysis revealed that Rfa2 levels were not recovered in the Rad53-S461D and Rad53-S545D mutants, similar to results obtained in the pph3Δ mutant (Fig. 6C). Hence, our results strongly suggest that failure of dephosphorylation at S461 and S545 on Rad53 may be responsible for the MMS-induced pseudohyphal growth and sensitivity in pph3Δ and psy2Δ mutants.

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