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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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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.
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pone-0037246-g004: 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.

Mentions: Northern blot (Fig. 3C) and qPCR (Fig. 4D) analyses produced consistent results in the expression levels of MSH2, RFA2, CCN1, PCL2 and HWP1, which are genes involved in either cell cycle regulation or hyphal growth. Among these genes, downregulation of RFA2 in pph3Δ and psy2Δ mutants after MMS recovery was further confirmed by Western blot analyses (Fig. 4D). Therefore, our results suggest that Rad53 hyperphosphorylation resulting from PPH3 and PSY2 deletion triggers its downstream signaling, which may contribute to MMS sensitivity.


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)

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.
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getmorefigures.php?uid=PMC3351423&req=5

pone-0037246-g004: 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.
Mentions: Northern blot (Fig. 3C) and qPCR (Fig. 4D) analyses produced consistent results in the expression levels of MSH2, RFA2, CCN1, PCL2 and HWP1, which are genes involved in either cell cycle regulation or hyphal growth. Among these genes, downregulation of RFA2 in pph3Δ and psy2Δ mutants after MMS recovery was further confirmed by Western blot analyses (Fig. 4D). Therefore, our results suggest that Rad53 hyperphosphorylation resulting from PPH3 and PSY2 deletion triggers its downstream signaling, which may contribute to MMS sensitivity.

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