Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351423&req=5

pone-0037246-g001: 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.

Mentions: Previous studies in Saccharomyces cerevisiae demonstrated that PPH3 deletion led to hypersensitivity towards MMS but not HU [34]. Thus, we first determined whether the same phenomenon also occurs in C. albicans. Wild-type, pph3Δ and psy2Δ yeast cells (Table 1) were inoculated into liquid YPD medium containing different concentrations of MMS or HU and incubated at 30°C for 6 h, followed by recovery in fresh drug-free YPD medium for 8 h at 30°C. Microscopic examination of the genotoxin-induced cell elongation at timed intervals revealed that both pph3Δ and psy2Δ mutants exhibited cell elongation during HU treatment and returned to the yeast form of growth after drug removal in manners comparable to wild-type cells (Fig. 1A, left & Fig. S2). In comparison, during MMS treatment the mutant cells exhibited faster elongation than wild-type cells and continued to elongate throughout the entire recovery period, while the wild-type cells returned to yeast growth ∼2 h after shifting to the drug free medium (Fig. 1A, right & Fig. S1).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351423&req=5

pone-0037246-g001: 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.
Mentions: Previous studies in Saccharomyces cerevisiae demonstrated that PPH3 deletion led to hypersensitivity towards MMS but not HU [34]. Thus, we first determined whether the same phenomenon also occurs in C. albicans. Wild-type, pph3Δ and psy2Δ yeast cells (Table 1) were inoculated into liquid YPD medium containing different concentrations of MMS or HU and incubated at 30°C for 6 h, followed by recovery in fresh drug-free YPD medium for 8 h at 30°C. Microscopic examination of the genotoxin-induced cell elongation at timed intervals revealed that both pph3Δ and psy2Δ mutants exhibited cell elongation during HU treatment and returned to the yeast form of growth after drug removal in manners comparable to wild-type cells (Fig. 1A, left & Fig. S2). In comparison, during MMS treatment the mutant cells exhibited faster elongation than wild-type cells and continued to elongate throughout the entire recovery period, while the wild-type cells returned to yeast growth ∼2 h after shifting to the drug free medium (Fig. 1A, right & Fig. S1).

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