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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 is targeted by the DNA damage response. (A) Total RNA was prepared from untreated (–) or HU-treated (+) cultures of wild-type (WT) and Yox1.SATA (SATA) cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (B) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation analysis of chromatin extracts isolated from untreated or HU-treated (10 mM HU, 4 h at 30°C) cultures of WT, SATA, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (C) The same chromatin extracts analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies. (D) Phosphorylation level of endogenous Chk1-HA in native extracts prepared from untreated (−) or HU-treated (+) cultures of WT, SATA, or ∆cds1 strains. Proteins were resolved in 8% SDS–PAGE and anti-HA Western blotted to detect Chk1.
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Figure 1: Cdc10 is targeted by the DNA damage response. (A) Total RNA was prepared from untreated (–) or HU-treated (+) cultures of wild-type (WT) and Yox1.SATA (SATA) cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (B) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation analysis of chromatin extracts isolated from untreated or HU-treated (10 mM HU, 4 h at 30°C) cultures of WT, SATA, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (C) The same chromatin extracts analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies. (D) Phosphorylation level of endogenous Chk1-HA in native extracts prepared from untreated (−) or HU-treated (+) cultures of WT, SATA, or ∆cds1 strains. Proteins were resolved in 8% SDS–PAGE and anti-HA Western blotted to detect Chk1.

Mentions: While investigating the effect of the DNA replication checkpoint on the regulation of the transcription factor MBF, we noticed that when cells were treated with hydroxyurea (HU) on a Yox1 mutant background (Yox1.SATA) that cannot be phosphorylated by the DNA replication checkpoint effector kinase Cds1, MBF-dependent induction of transcription was abrogated (Figure 1A; Gomez-Escoda et al., 2011). Under these conditions, the core MBF element, Cdc10, was released from chromatin (Figure 1B), in parallel to the release of the repressor Yox1 (Figure 1C). We were able to observe this release independent of the presence of Cds1, since in cells lacking Cds1, Cdc10 was also released when they were treated with HU and to a similar extent as in the Yox1.SATA cells. Unexpectedly, this release of Cdc10 was abrogated in the absence of Chk1 (both Δchk1 and Δchk1Δcds1 strains) or when Chk1 could not be activated in cells that lack the sensor kinase (Δrad3 strain). Of interest, we were able to observe that Chk1 was phosphorylated (which is a hallmark of its activation) when either Yox1.SATA or Δcds1 cells were treated with HU, pointing to the fact that in these specific genetic backgrounds both the DNA replication and DNA damage checkpoints were activated by HU (Figure 1D).


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 is targeted by the DNA damage response. (A) Total RNA was prepared from untreated (–) or HU-treated (+) cultures of wild-type (WT) and Yox1.SATA (SATA) cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (B) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation analysis of chromatin extracts isolated from untreated or HU-treated (10 mM HU, 4 h at 30°C) cultures of WT, SATA, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (C) The same chromatin extracts analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies. (D) Phosphorylation level of endogenous Chk1-HA in native extracts prepared from untreated (−) or HU-treated (+) cultures of WT, SATA, or ∆cds1 strains. Proteins were resolved in 8% SDS–PAGE and anti-HA Western blotted to detect Chk1.
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Related In: Results  -  Collection

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Figure 1: Cdc10 is targeted by the DNA damage response. (A) Total RNA was prepared from untreated (–) or HU-treated (+) cultures of wild-type (WT) and Yox1.SATA (SATA) cells and analyzed by hybridization to the probes indicated on the left. rRNA is shown as loading control. (B) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation analysis of chromatin extracts isolated from untreated or HU-treated (10 mM HU, 4 h at 30°C) cultures of WT, SATA, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (C) The same chromatin extracts analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies. (D) Phosphorylation level of endogenous Chk1-HA in native extracts prepared from untreated (−) or HU-treated (+) cultures of WT, SATA, or ∆cds1 strains. Proteins were resolved in 8% SDS–PAGE and anti-HA Western blotted to detect Chk1.
Mentions: While investigating the effect of the DNA replication checkpoint on the regulation of the transcription factor MBF, we noticed that when cells were treated with hydroxyurea (HU) on a Yox1 mutant background (Yox1.SATA) that cannot be phosphorylated by the DNA replication checkpoint effector kinase Cds1, MBF-dependent induction of transcription was abrogated (Figure 1A; Gomez-Escoda et al., 2011). Under these conditions, the core MBF element, Cdc10, was released from chromatin (Figure 1B), in parallel to the release of the repressor Yox1 (Figure 1C). We were able to observe this release independent of the presence of Cds1, since in cells lacking Cds1, Cdc10 was also released when they were treated with HU and to a similar extent as in the Yox1.SATA cells. Unexpectedly, this release of Cdc10 was abrogated in the absence of Chk1 (both Δchk1 and Δchk1Δcds1 strains) or when Chk1 could not be activated in cells that lack the sensor kinase (Δrad3 strain). Of interest, we were able to observe that Chk1 was phosphorylated (which is a hallmark of its activation) when either Yox1.SATA or Δcds1 cells were treated with HU, pointing to the fact that in these specific genetic backgrounds both the DNA replication and DNA damage checkpoints were activated by HU (Figure 1D).

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