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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

LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae. (49/1039)

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.  (+info)

Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1. (50/1039)

Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.  (+info)

Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality. (51/1039)

In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.  (+info)

Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation. (52/1039)

Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. Here we show that the ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and that T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells.  (+info)

Caenorhabditis elegans Chk2-like gene is essential for meiosis but dispensable for DNA repair. (53/1039)

A Chk2-like gene was identified in the genome of Caenorhabditis elegans. The putative gene product, termed Ce-chk-2 consists of 450 amino acid residues, and shows good homology with the Chk2/Cds1 gene family. The results of RNA-mediated interference (RNAi) indicated that the F1 generation from dsRNA injected animals grew to adulthood, but approximately 95% of their eggs (F2) died during early embryogenesis. Among the few surviving progeny, males (XO animals) arose at an abnormally high frequency (30%). In addition, 12 univalents were observed in full grown oocytes of the F1, while six bivalents were normally observed in wild-type oocytes. Ce-chk-2 gene expression increased in the adult stage, and their expression level decreased in the glp-4 mutant, which is defective in germ line proliferation. The radiation sensitivity of F1 embryos carrying Ce-chk-2 RNAi was not significantly affected.  (+info)

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner. (54/1039)

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.  (+info)

The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. (55/1039)

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.  (+info)

Kinesin subfamily UNC104 contains a FHA domain: boundaries and physicochemical characterization. (56/1039)

By sequence analysis we show that the U104 domain found in the UNC104 subfamily of kinesins is a forkhead homology-associated domain (FHA). A combination of limited proteolysis, mass spectroscopy, and physicochemical analysis define this domain as a genuine autonomously folding domain. Our data show that the FHA domain is shorter than previously reported since the C-terminal alpha-helix is not part of its minimum core. Key amino acids postulated to recognize phosphorylated residues are conserved. These data suggest that the kinesin FHA domains are functional domains involved in protein-protein interactions regulated by phosphorylation.  (+info)