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Checkpoint Kinase 2Protein KinasesProtein-Serine-Threonine KinasesCell Cycle ProteinsDNA DamageAtaxia Telangiectasia Mutated ProteinsGenes, cdcG2 PhaseM Phase Cell Cycle CheckpointsMitosisCell CyclePhosphorylationCell Cycle CheckpointsS PhaseProtein Kinase Inhibitorscdc25 PhosphatasesPhosphatidylinositol 3-KinasesDNA ReplicationHydroxyureaSaccharomyces cerevisiae ProteinsCDC2 Protein KinaseSpindle ApparatusMAP Kinase Signaling SystemSchizosaccharomyces pombe ProteinsSchizosaccharomycesG2 Phase Cell Cycle CheckpointsMutationDNA-Binding ProteinsNuclear ProteinsIntracellular Signaling Peptides and ProteinsS Phase Cell Cycle CheckpointsKinetochoresTumor Suppressor ProteinsSaccharomyces cerevisiaeDNA RepairCalcium-Calmodulin-Dependent Protein KinasesSignal TransductionProtein-Tyrosine KinasesCell Line, Tumorsrc-Family KinasesNocodazoleHeLa CellsRadiation, IonizingCyclin-Dependent KinasesProtein BindingEnzyme ActivationTumor Suppressor Protein p53Cdc20 ProteinsMethyl MethanesulfonateProtein Kinase Cp38 Mitogen-Activated Protein KinasesApoptosisDNA Breaks, Double-StrandedAurora KinasesG1 PhaseRNA, Small InterferingCyclic AMP-Dependent Protein KinasesModels, BiologicalMolecular Sequence DataFungal ProteinsUltraviolet RaysCell LineCyclin BMitogen-Activated Protein Kinase 1Chromosome SegregationDNA, FungalHistonesGenomic InstabilityAmino Acid SequenceMitogen-Activated Protein Kinase KinasesProtein Structure, TertiaryJNK Mitogen-Activated Protein Kinasesp21-Activated KinasesRNA InterferenceBlotting, WesternMitogen-Activated Protein Kinase 3Cell DivisionGamma RaysSerineStaurosporineReplication Protein AMAP Kinase Kinase KinasesCell SurvivalCreatine KinaseTransfectionCasein Kinase IICell NucleusMeiosisCDC2-CDC28 KinasesChromosomal Proteins, Non-HistoneMAP Kinase Kinase 1eIF-2 KinasePhosphoproteinsImmunoblottingGene Expression Regulation, FungalCasein KinasesRecombinant Fusion ProteinsPhosphoserineEnzyme InhibitorsPyruvate Kinase

Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. (65/1039)

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.  (+info)

Replication checkpoint: preventing mitotic catastrophe. (66/1039)

A conserved network of signal transduction pathways prevents mitosis if DNA is damaged or its synthesis incomplete. Loss of this checkpoint control is detrimental to the developing embryo. Recent studies have shed new light on how the essential ATR and Chk1 protein kinases cooperate to prevent such a crisis.  (+info)

Telomerase subunit overexpression suppresses telomere-specific checkpoint activation in the yeast yku80 mutant. (67/1039)

Ku is a conserved heterodimeric DNA-binding protein that plays critical roles in DNA repair and telomere homeostasis. In Saccharomyces cerevisiae, deletion of YKU70 or YKU80 results in an inability to grow at 37 degrees C. This is suppressed by overexpression of several components of telomerase (EST1, EST2 and TLC1). We show that overexpression of EST2 or TLC1 in yku80 mutants does not restore efficient DNA repair, or restore normal telomere function, as measured by telomere length, single-stranded G-rich strand or transcriptional silencing. Instead, yku80 mutants activate a Rad53p-dependent DNA-damage checkpoint at 37 degrees C and this is suppressed by overexpression of EST2 or TLC1. Indeed, deletion of genes required for Rad53p activation also suppresses the yku80 temperature sensitivity. These results suggest that activation of the DNA-damage checkpoint in yku mutants at 37 degrees C does not result from reduced telomere length per se, but reflects an alteration of the telomere structure that is recognized as damaged DNA.  (+info)

Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase. (68/1039)

Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1).  (+info)

Inhibition of the G2 DNA damage checkpoint and of protein kinases Chk1 and Chk2 by the marine sponge alkaloid debromohymenialdisine. (69/1039)

Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G(2) phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G(2) checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G(2) checkpoint inhibitors. It inhibited the G(2) checkpoint with an IC(50) of 8 micrometer and showed moderate cytotoxicity (IC(50) = 25 micrometer) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC(50) = 3 micrometer) and Chk2 (IC(50) = 3.5 micrometer) but not ataxia-telangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.  (+info)

Threonine-11, phosphorylated by Rad3 and atm in vitro, is required for activation of fission yeast checkpoint kinase Cds1. (70/1039)

Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T(11)Q(12). Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1(-) null mutant. Cds1(T11A) was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.  (+info)

Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants. (71/1039)

The antitumor drug adozelesin is a potent cytotoxic DNA-damaging agent. Here we determined how adozelesin affects chromosomal DNA replication at a molecular level in a yeast model system and examined the influence of checkpoint kinase genes, the human homologues of which are mutated in cancer. Analysis of replication intermediates using two-dimensional gel electrophoresis showed that adozelesin inhibited the activity of a replication origin and stalled replication fork progression through chromosomal DNA at the origin. RAD53 and MEC1 protein kinase genes, homologues of human CHK2 and ATM, respectively, regulate an intra-S-phase DNA damage checkpoint and, when mutated, permit unchecked replication of damaged DNA in S-phase. Mutations in these genes did not abrogate adozelesin-induced inhibition of origin activity and fork progression at the replication origin. However, novel replication intermediates indicative of DNA breaks were detected only in the rad53 mutant, suggesting a role for the wild-type gene in maintaining chromosome integrity in the presence of the drug. In contrast to the inhibition of the active replication origin by adozelesin, normally silent origins present in the same chromosome were activated by adozelesin in rad53 and mec1 mutant cells. Thus, an antitumor drug that damages DNA can induce an abnormal replication pattern in a chromosome by activating silent origins, depending upon defects in yeast checkpoint kinase genes, the homologues of which are mutated in cancer. Implications of an abnormal replication pattern for the epigenetic regulation of gene expression are discussed.  (+info)

Asf1 links Rad53 to control of chromatin assembly. (72/1039)

Yeast defective in the checkpoint kinase Rad53 fail to recover from transient DNA replication blocks and synthesize intact chromosomes. The effectors of Rad53 relevant to this recovery process are unknown. Here we report that overproduction of the chromatin assembly factor Asf1 can suppress the Ts phenotype of mrc1rad53 double mutants and the HU sensitivity of rad53 mutants. Eliminating silencing also suppresses this lethality, further implicating chromatin structure in checkpoint function. We find that Asf1 and Rad53 exist in a dynamic complex that dissociates in response to replication blocks and DNA damage. Thus, checkpoint pathways directly regulate chromatin assembly to promote survival in response to DNA damage and replication blocks.  (+info)