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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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The DNA damage response releases intact MBF from its target promoters. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation (ChIP) analysis with anti-Cdc10 polyclonal antibodies of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of WT strain (left) or from irradiated cells (100 Gy; right). (B) The interaction between Cdc10 and Res2 is preserved in MMS-treated cells. Extracts (2.5 mg) from WT and Cdc10-HA strains (with or without MMS treatment for the time indicate on top) were immunoprecipitated with anti-HA antibody and analyzed for the presence of Res2 and Cdc10 with specific antibodies (monoclonal anti-Res2 and anti-HA, respectively). (C) Loading of Res1 (left) or Res2 (right) on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of a Res1-HA or Res2-HA strain, respectively.
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Figure 2: The DNA damage response releases intact MBF from its target promoters. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation (ChIP) analysis with anti-Cdc10 polyclonal antibodies of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of WT strain (left) or from irradiated cells (100 Gy; right). (B) The interaction between Cdc10 and Res2 is preserved in MMS-treated cells. Extracts (2.5 mg) from WT and Cdc10-HA strains (with or without MMS treatment for the time indicate on top) were immunoprecipitated with anti-HA antibody and analyzed for the presence of Res2 and Cdc10 with specific antibodies (monoclonal anti-Res2 and anti-HA, respectively). (C) Loading of Res1 (left) or Res2 (right) on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of a Res1-HA or Res2-HA strain, respectively.

Mentions: It was previously shown that, besides Yox1, other components of the MBF complex (Nrm1 and Cdc10) could be phosphorylated by Cds1 when cells were under replicative stress (de Bruin et al., 2008; Dutta et al., 2008). However, our previous results (Figure 1) pointed to the possibility that some MBF components could also be targeted by Chk1 specifically when the DNA damage checkpoint was activated. To further investigate the signaling from this checkpoint to the MBF factor, we treated fission yeast cells with different DNA-damaging agents, such as MMS and γ-irradiation (Figure 2A). Indeed, both damaging agents were able to induce the release of Cdc10 from two of the better-characterized MBF-dependent promoters, cdc18 and cdc22. To determine whether Cdc10 was released alone or with other components of the MBF complex, we decided to test for coimmunoprecipitation between Cdc10 and Res2, which contains the DNA-binding activity of the MBF complex in its amino-terminal region (Miyamoto et al., 1994; Obara-Ishihara and Okayama, 1994). As shown in Figure 2B, the interaction between Cdc10 and Res2 was well preserved, if not improved, after treating fission yeast cells with MMS. In fact, both Res1 and Res2 are released from chromatin after treatment with MMS (Figure 2C), pointing to the possibility that the core elements of MBF (Res1, Res2, and Cdc10) are released as a complex from chromatin (and not as individual components) after the DNA damage checkpoint is induced.


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)

The DNA damage response releases intact MBF from its target promoters. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation (ChIP) analysis with anti-Cdc10 polyclonal antibodies of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of WT strain (left) or from irradiated cells (100 Gy; right). (B) The interaction between Cdc10 and Res2 is preserved in MMS-treated cells. Extracts (2.5 mg) from WT and Cdc10-HA strains (with or without MMS treatment for the time indicate on top) were immunoprecipitated with anti-HA antibody and analyzed for the presence of Res2 and Cdc10 with specific antibodies (monoclonal anti-Res2 and anti-HA, respectively). (C) Loading of Res1 (left) or Res2 (right) on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of a Res1-HA or Res2-HA strain, respectively.
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Figure 2: The DNA damage response releases intact MBF from its target promoters. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by chromatin immunoprecipitation (ChIP) analysis with anti-Cdc10 polyclonal antibodies of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of WT strain (left) or from irradiated cells (100 Gy; right). (B) The interaction between Cdc10 and Res2 is preserved in MMS-treated cells. Extracts (2.5 mg) from WT and Cdc10-HA strains (with or without MMS treatment for the time indicate on top) were immunoprecipitated with anti-HA antibody and analyzed for the presence of Res2 and Cdc10 with specific antibodies (monoclonal anti-Res2 and anti-HA, respectively). (C) Loading of Res1 (left) or Res2 (right) on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or MMS-treated (0.1% for 1 h at 30ºC) cultures of a Res1-HA or Res2-HA strain, respectively.
Mentions: It was previously shown that, besides Yox1, other components of the MBF complex (Nrm1 and Cdc10) could be phosphorylated by Cds1 when cells were under replicative stress (de Bruin et al., 2008; Dutta et al., 2008). However, our previous results (Figure 1) pointed to the possibility that some MBF components could also be targeted by Chk1 specifically when the DNA damage checkpoint was activated. To further investigate the signaling from this checkpoint to the MBF factor, we treated fission yeast cells with different DNA-damaging agents, such as MMS and γ-irradiation (Figure 2A). Indeed, both damaging agents were able to induce the release of Cdc10 from two of the better-characterized MBF-dependent promoters, cdc18 and cdc22. To determine whether Cdc10 was released alone or with other components of the MBF complex, we decided to test for coimmunoprecipitation between Cdc10 and Res2, which contains the DNA-binding activity of the MBF complex in its amino-terminal region (Miyamoto et al., 1994; Obara-Ishihara and Okayama, 1994). As shown in Figure 2B, the interaction between Cdc10 and Res2 was well preserved, if not improved, after treating fission yeast cells with MMS. In fact, both Res1 and Res2 are released from chromatin after treatment with MMS (Figure 2C), pointing to the possibility that the core elements of MBF (Res1, Res2, and Cdc10) are released as a complex from chromatin (and not as individual components) after the DNA damage checkpoint is induced.

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