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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 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.
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pone-0037246-g003: 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.

Mentions: Results showed that there is an overall downregulation of MBF-regulated genes, such as MSH2, RFA2, CCN1 and PCL2, upon MMS treatment and during recovery in the pph3Δ or psy2Δ mutant as compared to wild-type cells (Fig. 3A&B). Upon MMS treatment, upregulation of MSH2, RFA2 and CCN1 was observed in wild-type, but to a minor level in the pph3Δ and psy2Δ mutant; while PCL2 was downregulated in all three strains (Fig. 3A–C). Higher than normal levels of MSH2, CCN1 and RFA2 persisted in wild-type cells after recovery, while RFA2 returned to normal levels in the pph3Δ and psy2Δ mutant during recovery (Fig. 3B&C). In contrast, PCL2 exhibited higher-than-normal expression levels after MMS recovery in wild-type cells, but exhibited near normal expression levels after recovery in the pph3Δ and psy2Δ mutant (Fig. 3B&C). Negative controls without reverse transcriptase was used in the RT-PCR experiments to rule out genomic DNA contamination during PCR amplification (Fig. 3A). GADPH was used as loading control, and rRNA was included to indicate RNA integrity in Northern blot experiment (Fig. 3B).


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351423&req=5

pone-0037246-g003: 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.
Mentions: Results showed that there is an overall downregulation of MBF-regulated genes, such as MSH2, RFA2, CCN1 and PCL2, upon MMS treatment and during recovery in the pph3Δ or psy2Δ mutant as compared to wild-type cells (Fig. 3A&B). Upon MMS treatment, upregulation of MSH2, RFA2 and CCN1 was observed in wild-type, but to a minor level in the pph3Δ and psy2Δ mutant; while PCL2 was downregulated in all three strains (Fig. 3A–C). Higher than normal levels of MSH2, CCN1 and RFA2 persisted in wild-type cells after recovery, while RFA2 returned to normal levels in the pph3Δ and psy2Δ mutant during recovery (Fig. 3B&C). In contrast, PCL2 exhibited higher-than-normal expression levels after MMS recovery in wild-type cells, but exhibited near normal expression levels after recovery in the pph3Δ and psy2Δ mutant (Fig. 3B&C). Negative controls without reverse transcriptase was used in the RT-PCR experiments to rule out genomic DNA contamination during PCR amplification (Fig. 3A). GADPH was used as loading control, and rRNA was included to indicate RNA integrity in Northern blot experiment (Fig. 3B).

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