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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are needed to regulate checkpoint protein kinases Chk1 and Cds1 in fission yeast. Chk1 is required to prevent mitosis when DNA is damaged by ionizing radiation (IR), whereas either kinase is sufficient to prevent mitosis when DNA replication is inhibited by hydroxyurea (HU). Checkpoint Rad proteins are required for IR-induced phosphorylation of Chk1 and HU-induced activation of Cds1. IR activates Cds1 only during the DNA synthesis (S) phase, whereas HU induces Chk1 phosphorylation only in cds1 mutants. Here, we investigate the basis of the checkpoint signal specificity of Chk1 phosphorylation and Cds1 activation. We show that IR fails to induce Chk1 phosphorylation in HU-arrested cells. Release from the HU arrest following IR causes substantial Chk1 phosphorylation. These and other data indicate that Cds1 prevents Chk1 phosphorylation in HU-arrested cells, which suggests that Cds1 actively suppresses a repair process that leads to Chk1 phosphorylation. Cds1 becomes more highly concentrated in the nucleus only during the S phase of the cell cycle. This finding correlates with S-phase specificity of IR-induced activation of Cds1. However, constitutive nuclear localization of Cds1 does not enhance IR-induced activation of Cds1. This result suggests that Cds1 activation requires DNA structures or protein activities that are present only during S phase. These findings help to explain how Chk1 and Cds1 respond to different checkpoint signals.
Mol Cell Biol 1999 Jun
PMID:Basis for the checkpoint signal specificity that regulates Chk1 and Cds1 protein kinases. 1033 Jan 67

Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase epsilon (Dpb11-Polepsilon complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polepsilon associated with these fragments. Association of Dpb11 and Polepsilon with these fragments was mutually dependent, suggesting that the Dpb11-Polepsilon complex associates with the ARS. Moreover, Dpb11 was required for the association of Polalpha-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polepsilon complex with the ARS fragments is required for the association of the Polalpha-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polepsilon in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases alpha and epsilon and the ARS.
Mol Cell Biol 2000 Apr
PMID:Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast. 1073 84

The S-M checkpoint is an intracellular signaling pathway that ensures that mitosis is not initiated in cells undergoing DNA replication. We identified cid1, a novel fission yeast gene, through its ability when overexpressed to confer specific resistance to a combination of hydroxyurea, which inhibits DNA replication, and caffeine, which overrides the S-M checkpoint. Cid1 overexpression also partially suppressed the hydroxyurea sensitivity characteristic of DNA polymerase delta mutants and mutants defective in the "checkpoint Rad" pathway. Cid1 is a member of a family of putative nucleotidyltransferases including budding yeast Trf4 and Trf5, and mutation of amino acid residues predicted to be essential for this activity resulted in loss of Cid1 function in vivo. Two additional Cid1-like proteins play similar but nonredundant checkpoint-signaling roles in fission yeast. Cells lacking Cid1 were found to be viable but specifically sensitive to the combination of hydroxyurea and caffeine and to be S-M checkpoint defective in the absence of Cds1. Genetic data suggest that Cid1 acts in association with Crb2/Rhp9 and through the checkpoint-signaling kinase Chk1 to inhibit unscheduled mitosis specifically when DNA polymerase delta or epsilon is inhibited.
Mol Cell Biol 2000 May
PMID:Cid1, a fission yeast protein required for S-M checkpoint control when DNA polymerase delta or epsilon is inactivated. 1075 7

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that Xenopus Cds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-free Xenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast, Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.
Mol Biol Cell 2000 May
PMID:Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends. 1079 33

Mitotic checkpoints restrain the onset of mitosis (M) when DNA is incompletely replicated or damaged. These checkpoints are conserved between the fission yeast Schizosaccharomyces pombe and mammals. In both types of organisms, the methylxanthine caffeine overrides the synthesis (S)-M checkpoint that couples mitosis to completion of DNA S phase. The molecular target of caffeine was sought in fission yeast. Caffeine prevented activation of Cds1 and phosphorylation of Chk1, two protein kinases that enforce the S-M checkpoint triggered by hydroxyurea. Caffeine did not inhibit these kinases in vitro but did inhibit Rad3, a kinase that regulates Cds1 and Chk1. In accordance with this finding, caffeine also overrode the G(2)-M DNA damage checkpoint that requires Rad3 function. Rad3 coprecipitated with Cds1 expressed at endogenous amounts, a finding that supports the hypothesis that Rad3 is involved in direct activation of Cds1.
Mol Cell Biol 2000 Jun
PMID:Mechanism of caffeine-induced checkpoint override in fission yeast. 1082 92

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells.
Mol Cell Biol 2000 Jul
PMID:Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest. 1084 81

To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations of Schizosaccharomyces pombe DNA primase. DNA primase has two subunits, Spp1 and Spp2 (S. pombe primases 1 and 2). Spp1 is the catalytic subunit that synthesizes the RNA primer, which is then extended by DNA polymerase alpha (Polalpha) to synthesize an initiation DNA structure, and this catalytic function of Polalpha is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Polalpha. Thermosensitive mutations of spp2(+) destabilize the Polalpha-primase complex, resulting in an allele-specific S phase checkpoint defect. The mutant exhibiting a more severe checkpoint defect also has a higher extent of Polalpha-primase complex instability and deficiency in the hydroxyurea-induced Cds1-mediated intra-S phase checkpoint response. However, this mutant is able to activate the Cds1 response to S phase arrest induced by temperature. These findings suggest that the Cds1 response to the S-phase arrest signal(s) induced by a initiation mutant is different from that induced by hydroxyurea. Interestingly, a polalphats mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Polalpha-primase complex stability, and the Cds1 response induced by hydroxyurea or by the mutant arrests at the restrictive temperature. Thus, the Cds1-mediated intra-S phase checkpoint response induced by hydroxyurea can also be distinguished from the S-M phase checkpoint response that requires the initiation DNA synthesis by Polalpha.
Mol Cell Biol 2000 Nov
PMID:Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation. 1102 57

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation and characterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis, hsk1(ts) genetically interacts with several S-phase mutants. At the restrictive temperature, hsk1(ts) cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest. Interestingly, hsk1(ts) mutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phase role, hsk1(ts) is synthetically lethal with Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.
Mol Cell Biol 2000 Nov
PMID:Schizosaccharomyces pombe Hsk1p is a potential cds1p target required for genome integrity. 1102 63

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.
Mol Cell Biol 2000 Dec
PMID:Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1. 1107 77

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.
Mol Cell Biol 2000 Dec
PMID:Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality. 1107 5


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