Antagonism of Chk1 Signaling in the G2 DNA Damage Checkpoint by Dominant Alleles of Cdr1
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Activation of the Chk1 protein kinase by DNA damage enforces a checkpoint that maintains Cdc2 in its inactive, tyrosine-15 (Y15) phosphorylated state. Chk1 downregulates the Cdc25 phosphatases and concomitantly upregulates the Wee1 kinases that control the phosphorylation of Cdc2. Overproduction of Chk1 causes G(2) arrest/delay independently of DNA damage and upstream checkpoint genes. We utilized this to screen fission yeast for mutations that alter sensitivity to Chk1 signaling. We describe three dominant-negative alleles of cdr1, which render cells supersensitive to Chk1 levels, and suppress the checkpoint defects of chk1Delta cells. Cdr1 encodes a protein kinase previously identified as a negative regulator of Wee1 activity in response to limited nutrition, but Cdr1 has not previously been linked to checkpoint signaling. Overproduction of Cdr1 promotes checkpoint defects and exacerbates the defective response to DNA damage of cells lacking Chk1. We conclude that regulation of Wee1 by Cdr1 and possibly by related kinases is an important antagonist of Chk1 signaling and represents a novel negative regulation of cell cycle arrest promoted by this checkpoint.Keywords:
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The protein kinase Chk1 is essential for the DNA damage checkpoint. Cells lacking Chk1 are hypersensitive to DNA-damaging agents such as UV light and gamma-irradiation because they fail to arrest the cell cycle when DNA damage is generated. Phosphorylation of Chk1 occurs after DNA damage and is dependent on the integrity of the DNA damage checkpoint pathway. We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA damage in the fission yeast Schizosaccharomyces pombe that results in Chk1 phosphorylation. We demonstrate that Chk1 is phosphorylated in response to CPT treatment in a time- and dose-dependent manner and that phosphorylation is dependent on an intact DNA damage checkpoint pathway. Furthermore, we show that cells must be actively dividing in order for CPT to generate a Chk1-responsive DNA damage signal. This observation is consistent with a model whereby the cytotoxic event caused by CPT treatment is the production of a DNA double-strand break resulting from the collision of a DNA replication fork with a trapped CPT-topoisomerase I cleavable complex. Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpoint pathway can be an important determinant for CPT sensitivity or resistance. Finally, as a well-characterized, soluble agent that specifically causes DNA damage, CPT will allow a biochemical analysis of the checkpoint pathway that responds to DNA damage.
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801 DNA damage checkpoints are regulatory signal transduction pathways that allow cells to control DNA damage-induced cell cycle arrest, DNA repair, and cell death by apoptosis. Most human tumors arise from multiple genetic changes, including defective DNA damage checkpoints that provide potential targets for therapeutic use of DNA-damaging agents. One strategy to enhance the impact of these agents is to inhibit the checkpoint signaling in cancer cells. The checkpoint kinase 1 (Chk1) is one of the effector kinases that regulates cell cycle progression after DNA damage. Upon DNA damage, Chk1 is phosphorylated on Ser 345 and 317, and this phosphorylation is dependent on ATM (ataxia-telangiectasia mutated) or ATR (ATM and Rad 3-related). In addition to phosphorylation, DNA damage-dependent Chk1 activity could be modulated by other posttranslational modifications and protein-protein interaction. We have developed experimental procedures for identification of novel Chk1 posttranslational modifications and associated proteins. Using immunoprecipitation followed by mass spectroscopy analysis, we have found novel candidate Chk1-associated proteins. Potentially, interaction of these proteins with Chk1 could inhibit DNA damage-dependent Chk1 activation, leading to abrogation of checkpoint response and sensitization of cancer cells to DNA-damaging drugs.
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MTA1 (metastasis-associated protein 1), an integral component of the nucleosome remodeling and deacetylase complex, has recently been implicated in the ionizing radiation-induced DNA damage response. However, whether MTA1 also participates in the UV-induced DNA damage checkpoint pathway remains unknown. In response to UV radiation, ATR (ataxia teleangiectasia- and Rad3-related) is the major kinase activated that orchestrates cell cycle progression with DNA repair machinery by phosphorylating and activating a number of downstream substrates, such as Chk1 (checkpoint kinase 1) and H2AX (histone 2A variant X). Here, we report that UV radiation stabilizes MTA1 in an ATR-dependent manner and increases MTA1 binding to ATR. On the other hand, depletion of MTA1 compromises the ATR-mediated Chk1 activation following UV treatment, accompanied by a marked down-regulation of Chk1 and its interacting partner Claspin, an adaptor protein that is required for the phosphorylation and activation of Chk1 by ATR. Furthermore, MTA1 deficiency decreases the induction of phosphorylated H2AX (referred to as gamma-H2AX) and gamma-H2AX focus formation after UV treatment. Consequently, depletion of MTA1 results in a defect in the G(2)-M checkpoint and increases cellular sensitivity to UV-induced DNA damage. Thus, MTA1 is required for the activation of the ATR-Claspin-Chk1 and ATR-H2AX pathways following UV treatment, and the noted abrogation of the DNA damage checkpoint in the MTA1-depleted cells may be, at least in part, a consequence of dysregulation of the expression of these two pathways. These findings suggest that, in addition to its role in the repair of double strand breaks caused by ionizing radiation, MTA1 also participates in the UV-induced ATR-mediated DNA damage checkpoint pathway.
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On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.
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Eukaryotic cells have evolved surveillance mechanisms, known as DNA-damage checkpoints, that sense and respond to genome damage. DNA-damage checkpoint pathways ensure co-ordinated cellular responses to DNA damage, including cell cycle delays and activation of repair mechanisms. RAD9, from Saccharomyces cerevisiae, was the first damage checkpoint gene to be identified, although its biochemical function remained unknown until recently. This review examines briefly work that provides significant insight into how Rad9 activates the checkpoint signalling kinase Rad53.
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DNA Damage Repair
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Deletion of the Saccharomyces cerevisiae TOP3gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391–8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Δ strains. We show that top3Δ mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Δ strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Δ mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.
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Methyl methanesulfonate
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S phase
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The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by “sensing” DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1–GFP or Ddc2–GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2–GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.
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A damage is a general event in the life of cells and may lead to mutation, cancer, and cell/organ death. DNA damage occurring in different phases of cell cycle can activate different damage checkpoint pathways to halt the progress of cell cycle in order to provide time for DNA damage repair. If DNA damage cannot be repaired, cellular apoptosis may be induced. Therefore, DNA damage checkpoint is of great significance for cell survival after DNA damage. This article summarizes recent research on DNA damage responses, including DNA damage checkpoint, DNA damage repair, transcriptional response, and cell apoptosis. We focus on how the DNA damage checkpoint pathway is activated after DNA damage, as well as the functional mechanism of the DNA damage checkpoint pathway. The review aims to help readers understand the great significance of DNA damage checkpoint pathway, providing a theoretical basis for its application in radiotherapy and chemotherapy for cancer.
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DNA damage; Checkpoint pathway; Cell cycle; Signal transduction; Cell apoptosis
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The protein kinase Chk1 is essential for the DNA damage checkpoint. Cells lacking Chk1 are hypersensitive to DNA-damaging agents such as UV light and γ-irradiation because they fail to arrest the cell cycle when DNA damage is generated. Phosphorylation of Chk1 occurs after DNA damage and is dependent on the integrity of the DNA damage checkpoint pathway. We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA damage in the fission yeast Schizosaccharomyces pombe that results in Chk1 phosphorylation. We demonstrate that Chk1 is phosphorylated in response to CPT treatment in a time- and dose-dependent manner and that phosphorylation is dependent on an intact DNA damage checkpoint pathway. Furthermore, we show that cells must be actively dividing in order for CPT to generate a Chk1-responsive DNA damage signal. This observation is consistent with a model whereby the cytotoxic event caused by CPT treatment is the production of a DNA double-strand break resulting from the collision of a DNA replication fork with a trapped CPT-topoisomerase I cleavable complex. Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpoint pathway can be an important determinant for CPT sensitivity or resistance. Finally, as a well-characterized, soluble agent that specifically causes DNA damage, CPT will allow a biochemical analysis of the checkpoint pathway that responds to DNA damage. Copyright © 1999 John Wiley & Sons, Ltd.
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Camptothecin
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On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop. Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle. The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12. Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.
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DNA re-replication
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