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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 Ser-720 and Ser-732 are phosphorylated by Chk1, inactivating MBF-dependent transcription. (A) Chk1 signals MBF through the C-terminal region of Cdc10. Loading of Cdc10 on cdc22 and cdc18 promoters was measured in untreated or MMS-treated (0.1% MMS, 1 h at 25ºC) cultures of WT and cdc10-C4 strain by ChIP. Average of three individual experiments (±SD). (B) Amino acid sequence of the Cdc10 region phosphorylated by Chk1. The phosphorylation consensus is indicated at the bottom. (C) Chk1 in vitro kinase activity (in arbitrary units) was assayed using GST, WT, Cdc10, or the Cdc10 mutants indicated on top as substrates. Coomassie staining of the gel is shown at the bottom. (D) Cdc10 phosphorylation was determined on extracts prepared from untreated (unt.) or MMS-treated (0.1%) cells from WT (Cdc10) or Cdc10.2A strains. Immunoprecipitates were analyzed by Western blot with anti-phosphoserine (α-P-Ser) or anti-Cdc10 antibodies (α-Cdc10). (E) Extracts from the tagged strains indicated on top, untreated (unt) or treated with 0.1% MMS for 60 min, were immunoprecipitated with anti-Myc antibody and analyzed for the presence of Chk1 and Cdc10 with specific antibodies (HA and Myc, respectively). Left, Western blot of the whole-cell extracts used in the immunoprecipitations.
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Figure 5: Cdc10 Ser-720 and Ser-732 are phosphorylated by Chk1, inactivating MBF-dependent transcription. (A) Chk1 signals MBF through the C-terminal region of Cdc10. Loading of Cdc10 on cdc22 and cdc18 promoters was measured in untreated or MMS-treated (0.1% MMS, 1 h at 25ºC) cultures of WT and cdc10-C4 strain by ChIP. Average of three individual experiments (±SD). (B) Amino acid sequence of the Cdc10 region phosphorylated by Chk1. The phosphorylation consensus is indicated at the bottom. (C) Chk1 in vitro kinase activity (in arbitrary units) was assayed using GST, WT, Cdc10, or the Cdc10 mutants indicated on top as substrates. Coomassie staining of the gel is shown at the bottom. (D) Cdc10 phosphorylation was determined on extracts prepared from untreated (unt.) or MMS-treated (0.1%) cells from WT (Cdc10) or Cdc10.2A strains. Immunoprecipitates were analyzed by Western blot with anti-phosphoserine (α-P-Ser) or anti-Cdc10 antibodies (α-Cdc10). (E) Extracts from the tagged strains indicated on top, untreated (unt) or treated with 0.1% MMS for 60 min, were immunoprecipitated with anti-Myc antibody and analyzed for the presence of Chk1 and Cdc10 with specific antibodies (HA and Myc, respectively). Left, Western blot of the whole-cell extracts used in the immunoprecipitations.

Mentions: Cdc10 has four putative sites that can be phosphorylated by Chk1 (Ser-563, Thr-603, Ser-720, and Ser-732). Whereas the first two are in close proximity to the ankyrin domain, which mediates protein–protein interactions, the last two residues are in the C-terminal region of Cdc10, which is essential for loading the Yox1/Nrm1 repressor system onto chromatin (Supplemental Figure S2). In fact, and as a first approach, we noticed that Cdc10 release after MMS treatment was not observed in a strain that lacks the last 61 amino acids of Cdc10, cdc10-C4 (Figure 5A). Thus we decided to focus on this carboxy-terminal domain of Cdc10 as a potential substrate of Chk1 phosphorylation. In fact, in our in vitro Chk1 kinase assays, a Cdc10 construct lacking the last 61 amino acids was not phosphorylated. Conversely, a construct containing only the carboxy-terminal 61 amino acids (and thus containing the last two putative phosphorylation sites) was consistently phosphorylated (Figure 5, B and C). When Ser-720 or Ser-732 was mutated to alanine, the extent of phosphorylation was diminished. Furthermore, in the double mutant, Cdc10 phosphorylation by Chk1 was completely abolished (Figure 5C). It is worth noting that these phosphorylation sites are partially different from the described as Cds1-phosphorylation sites in Cdc10, where the authors noticed that only when both Ser-720 and Thr-723 were mutated to glutamic acid was MBF-dependent transcription induced (Dutta et al., 2008). In fact, in this mutant background, the Nrm1/Yox1 repressor system is unable to bind the MBF complex (unpublished data). Next, to determine whether in vivo Chk1 was able to phosphorylate Cdc10 on Ser-720 and Ser-732, we used an anti-phosphoserine antibody. As shown in Figure 5D, Cdc10 is phosphorylated when cells are treated with MMS. However, when Ser-720 and Ser-732 were replaced by alanines, we were unable to detect this phosphorylation. In fact, and confirming the notion that the DNA damage checkpoint could be regulating the MBF complex, we were able to detect direct interaction between Chk1 and Cdc10 by coimmunoprecipitation (Figure 5E)


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 Ser-720 and Ser-732 are phosphorylated by Chk1, inactivating MBF-dependent transcription. (A) Chk1 signals MBF through the C-terminal region of Cdc10. Loading of Cdc10 on cdc22 and cdc18 promoters was measured in untreated or MMS-treated (0.1% MMS, 1 h at 25ºC) cultures of WT and cdc10-C4 strain by ChIP. Average of three individual experiments (±SD). (B) Amino acid sequence of the Cdc10 region phosphorylated by Chk1. The phosphorylation consensus is indicated at the bottom. (C) Chk1 in vitro kinase activity (in arbitrary units) was assayed using GST, WT, Cdc10, or the Cdc10 mutants indicated on top as substrates. Coomassie staining of the gel is shown at the bottom. (D) Cdc10 phosphorylation was determined on extracts prepared from untreated (unt.) or MMS-treated (0.1%) cells from WT (Cdc10) or Cdc10.2A strains. Immunoprecipitates were analyzed by Western blot with anti-phosphoserine (α-P-Ser) or anti-Cdc10 antibodies (α-Cdc10). (E) Extracts from the tagged strains indicated on top, untreated (unt) or treated with 0.1% MMS for 60 min, were immunoprecipitated with anti-Myc antibody and analyzed for the presence of Chk1 and Cdc10 with specific antibodies (HA and Myc, respectively). Left, Western blot of the whole-cell extracts used in the immunoprecipitations.
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Figure 5: Cdc10 Ser-720 and Ser-732 are phosphorylated by Chk1, inactivating MBF-dependent transcription. (A) Chk1 signals MBF through the C-terminal region of Cdc10. Loading of Cdc10 on cdc22 and cdc18 promoters was measured in untreated or MMS-treated (0.1% MMS, 1 h at 25ºC) cultures of WT and cdc10-C4 strain by ChIP. Average of three individual experiments (±SD). (B) Amino acid sequence of the Cdc10 region phosphorylated by Chk1. The phosphorylation consensus is indicated at the bottom. (C) Chk1 in vitro kinase activity (in arbitrary units) was assayed using GST, WT, Cdc10, or the Cdc10 mutants indicated on top as substrates. Coomassie staining of the gel is shown at the bottom. (D) Cdc10 phosphorylation was determined on extracts prepared from untreated (unt.) or MMS-treated (0.1%) cells from WT (Cdc10) or Cdc10.2A strains. Immunoprecipitates were analyzed by Western blot with anti-phosphoserine (α-P-Ser) or anti-Cdc10 antibodies (α-Cdc10). (E) Extracts from the tagged strains indicated on top, untreated (unt) or treated with 0.1% MMS for 60 min, were immunoprecipitated with anti-Myc antibody and analyzed for the presence of Chk1 and Cdc10 with specific antibodies (HA and Myc, respectively). Left, Western blot of the whole-cell extracts used in the immunoprecipitations.
Mentions: Cdc10 has four putative sites that can be phosphorylated by Chk1 (Ser-563, Thr-603, Ser-720, and Ser-732). Whereas the first two are in close proximity to the ankyrin domain, which mediates protein–protein interactions, the last two residues are in the C-terminal region of Cdc10, which is essential for loading the Yox1/Nrm1 repressor system onto chromatin (Supplemental Figure S2). In fact, and as a first approach, we noticed that Cdc10 release after MMS treatment was not observed in a strain that lacks the last 61 amino acids of Cdc10, cdc10-C4 (Figure 5A). Thus we decided to focus on this carboxy-terminal domain of Cdc10 as a potential substrate of Chk1 phosphorylation. In fact, in our in vitro Chk1 kinase assays, a Cdc10 construct lacking the last 61 amino acids was not phosphorylated. Conversely, a construct containing only the carboxy-terminal 61 amino acids (and thus containing the last two putative phosphorylation sites) was consistently phosphorylated (Figure 5, B and C). When Ser-720 or Ser-732 was mutated to alanine, the extent of phosphorylation was diminished. Furthermore, in the double mutant, Cdc10 phosphorylation by Chk1 was completely abolished (Figure 5C). It is worth noting that these phosphorylation sites are partially different from the described as Cds1-phosphorylation sites in Cdc10, where the authors noticed that only when both Ser-720 and Thr-723 were mutated to glutamic acid was MBF-dependent transcription induced (Dutta et al., 2008). In fact, in this mutant background, the Nrm1/Yox1 repressor system is unable to bind the MBF complex (unpublished data). Next, to determine whether in vivo Chk1 was able to phosphorylate Cdc10 on Ser-720 and Ser-732, we used an anti-phosphoserine antibody. As shown in Figure 5D, Cdc10 is phosphorylated when cells are treated with MMS. However, when Ser-720 and Ser-732 were replaced by alanines, we were unable to detect this phosphorylation. In fact, and confirming the notion that the DNA damage checkpoint could be regulating the MBF complex, we were able to detect direct interaction between Chk1 and Cdc10 by coimmunoprecipitation (Figure 5E)

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