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The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

Ivanova T, Alves-Rodrigues I, Gómez-Escoda B, Dutta C, DeCaprio JA, Rhind N, Hidalgo E, Ayté J - Mol. Biol. Cell (2013)

Bottom Line: We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription.This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin.This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona 08003, Spain Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.

ABSTRACT
In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

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Cdc10 phosphorylation after DNA damage is essential for viability. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or treated (0.1% MMS, 1 h at 30ºC) cultures of WT Cdc10 or the mutants indicated at the bottom. (B) Phosphorylation of S720 and S732 after ionizing radiation (IR) induces the release of Cdc10 from chromatin. Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or IR (100 Gy) cultures of WT and Cdc10.2A cells. Average of three individual experiments (±SD). (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of a cdc10.2AΔyox1Δnrm1 strain and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) RNA was prepared from wild-type (Cdc10) or Cdc10.2A cells exponentially growing or treated with 0.1% MMS for 1 h. cdc18, cdt2, cdc22, and mik1 were quantitated by RT-qPCR. Results are shown as fold induction over untreated wild-type cells as the average of three individual experiments (±SD). (E) Survival was performed by spotting 10–105 cells of the indicated strains (in a Δyox1Δnrm1 background) onto YE5S plates in the absence or presence of MMS or HU. Plates were incubated at 30°C for 3–4 d.
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Figure 6: Cdc10 phosphorylation after DNA damage is essential for viability. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or treated (0.1% MMS, 1 h at 30ºC) cultures of WT Cdc10 or the mutants indicated at the bottom. (B) Phosphorylation of S720 and S732 after ionizing radiation (IR) induces the release of Cdc10 from chromatin. Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or IR (100 Gy) cultures of WT and Cdc10.2A cells. Average of three individual experiments (±SD). (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of a cdc10.2AΔyox1Δnrm1 strain and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) RNA was prepared from wild-type (Cdc10) or Cdc10.2A cells exponentially growing or treated with 0.1% MMS for 1 h. cdc18, cdt2, cdc22, and mik1 were quantitated by RT-qPCR. Results are shown as fold induction over untreated wild-type cells as the average of three individual experiments (±SD). (E) Survival was performed by spotting 10–105 cells of the indicated strains (in a Δyox1Δnrm1 background) onto YE5S plates in the absence or presence of MMS or HU. Plates were incubated at 30°C for 3–4 d.

Mentions: To test whether Cdc10 phosphorylation by Chk1 is essential for in vivo regulation of Cdc10/MBF binding to its target promoters upon activation of the DNA damage checkpoint, we introduced serine-to-alanine mutations in fission yeast, replacing the endogenous copy of cdc10. When treated with MMS, the strains that carry single mutations (including those next to the ankyrin domain) responded in a similar manner to a wild-type strain, that is, Cdc10 was released from its target promoters (Figure 6A and Supplemental Figure S3). However, in a strain that carries the double mutation S720AS732A (here Cdc10.2A) and cannot be phosphorylated in vitro and in vivo by Chk1, the release of Cdc10 was impaired from cdc18 promoter after treatment with MMS (Figure 6A). Of interest, we could observe only a small effect on the regulation of its binding activity to cdc22 promoter, pointing to the fact that Chk1 might differentially regulate the binding of Cdc10 to only a subset of MBF-dependent genes. A similar effect was observed when cells were irradiated (Figure 6B).


The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

Ivanova T, Alves-Rodrigues I, Gómez-Escoda B, Dutta C, DeCaprio JA, Rhind N, Hidalgo E, Ayté J - Mol. Biol. Cell (2013)

Cdc10 phosphorylation after DNA damage is essential for viability. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or treated (0.1% MMS, 1 h at 30ºC) cultures of WT Cdc10 or the mutants indicated at the bottom. (B) Phosphorylation of S720 and S732 after ionizing radiation (IR) induces the release of Cdc10 from chromatin. Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or IR (100 Gy) cultures of WT and Cdc10.2A cells. Average of three individual experiments (±SD). (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of a cdc10.2AΔyox1Δnrm1 strain and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) RNA was prepared from wild-type (Cdc10) or Cdc10.2A cells exponentially growing or treated with 0.1% MMS for 1 h. cdc18, cdt2, cdc22, and mik1 were quantitated by RT-qPCR. Results are shown as fold induction over untreated wild-type cells as the average of three individual experiments (±SD). (E) Survival was performed by spotting 10–105 cells of the indicated strains (in a Δyox1Δnrm1 background) onto YE5S plates in the absence or presence of MMS or HU. Plates were incubated at 30°C for 3–4 d.
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Figure 6: Cdc10 phosphorylation after DNA damage is essential for viability. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or treated (0.1% MMS, 1 h at 30ºC) cultures of WT Cdc10 or the mutants indicated at the bottom. (B) Phosphorylation of S720 and S732 after ionizing radiation (IR) induces the release of Cdc10 from chromatin. Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or IR (100 Gy) cultures of WT and Cdc10.2A cells. Average of three individual experiments (±SD). (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of a cdc10.2AΔyox1Δnrm1 strain and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) RNA was prepared from wild-type (Cdc10) or Cdc10.2A cells exponentially growing or treated with 0.1% MMS for 1 h. cdc18, cdt2, cdc22, and mik1 were quantitated by RT-qPCR. Results are shown as fold induction over untreated wild-type cells as the average of three individual experiments (±SD). (E) Survival was performed by spotting 10–105 cells of the indicated strains (in a Δyox1Δnrm1 background) onto YE5S plates in the absence or presence of MMS or HU. Plates were incubated at 30°C for 3–4 d.
Mentions: To test whether Cdc10 phosphorylation by Chk1 is essential for in vivo regulation of Cdc10/MBF binding to its target promoters upon activation of the DNA damage checkpoint, we introduced serine-to-alanine mutations in fission yeast, replacing the endogenous copy of cdc10. When treated with MMS, the strains that carry single mutations (including those next to the ankyrin domain) responded in a similar manner to a wild-type strain, that is, Cdc10 was released from its target promoters (Figure 6A and Supplemental Figure S3). However, in a strain that carries the double mutation S720AS732A (here Cdc10.2A) and cannot be phosphorylated in vitro and in vivo by Chk1, the release of Cdc10 was impaired from cdc18 promoter after treatment with MMS (Figure 6A). Of interest, we could observe only a small effect on the regulation of its binding activity to cdc22 promoter, pointing to the fact that Chk1 might differentially regulate the binding of Cdc10 to only a subset of MBF-dependent genes. A similar effect was observed when cells were irradiated (Figure 6B).

Bottom Line: We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription.This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin.This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, Barcelona 08003, Spain Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.

ABSTRACT
In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

Show MeSH
Related in: MedlinePlus