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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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Related in: MedlinePlus

Cdc10 is released from chromatin upon DNA damage. (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, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (B) The same chromatin extracts were analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies.
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Figure 4: Cdc10 is released from chromatin upon DNA damage. (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, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (B) The same chromatin extracts were analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies.

Mentions: Next we wanted to further characterize the signaling from the DNA damage checkpoint to the MBF complex. To confirm that release of Cdc10 was exclusively due to activation of the DNA damage checkpoint (and that the DNA replication checkpoint was not involved in this release), we analyzed the binding of Cdc10 and Yox1 to cdc18 and cdc22 promoters in cells with impaired signaling in each or both of these checkpoint-signaling pathways after treatment with MMS. As shown in Figure 4A, the release of Cdc10 in cells lacking Cds1 was similar to that in wild-type cells. However, in cells lacking either Chk1 or the upstream activating kinase, Rad3, Cdc10 was not released after treatment with MMS. Under these concentrations of MMS, Yox1 release from chromatin paralleled the release of Cdc10, pointing to the possibility that it was the MBF complex as a whole that was released from chromatin when only the DNA damage checkpoint was induced (Figure 4B).


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 released from chromatin upon DNA damage. (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, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (B) The same chromatin extracts were analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies.
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Related In: Results  -  Collection

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Figure 4: Cdc10 is released from chromatin upon DNA damage. (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, ∆cds1, ∆chk1, ∆cds1∆chk1, or ∆rad3 cells. Endogenous Cdc10 is HA tagged, and the levels of binding are quantified on anti-HA immunoprecipitated DNA. (B) The same chromatin extracts were analyzed for Yox1 binding with anti-Yox1 polyclonal antibodies.
Mentions: Next we wanted to further characterize the signaling from the DNA damage checkpoint to the MBF complex. To confirm that release of Cdc10 was exclusively due to activation of the DNA damage checkpoint (and that the DNA replication checkpoint was not involved in this release), we analyzed the binding of Cdc10 and Yox1 to cdc18 and cdc22 promoters in cells with impaired signaling in each or both of these checkpoint-signaling pathways after treatment with MMS. As shown in Figure 4A, the release of Cdc10 in cells lacking Cds1 was similar to that in wild-type cells. However, in cells lacking either Chk1 or the upstream activating kinase, Rad3, Cdc10 was not released after treatment with MMS. Under these concentrations of MMS, Yox1 release from chromatin paralleled the release of Cdc10, pointing to the possibility that it was the MBF complex as a whole that was released from chromatin when only the DNA damage checkpoint was induced (Figure 4B).

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