Caffeine can override the S-M checkpoint in fission yeast

1999 ◽  
Vol 112 (6) ◽  
pp. 927-937 ◽  
Author(s):  
S.W. Wang ◽  
C. Norbury ◽  
A.L. Harris ◽  
T. Toda
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
S Phase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Control ◽  
Animal Cells ◽  
Replication Checkpoint ◽  
Phase Arrest ◽  
S Phase Arrest

The replication checkpoint (or ‘S-M checkpoint’) control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control.

scholarly journals Modulation of Cell Cycle–specific Gene Expressions at the Onset of S Phase Arrest Contributes to the Robust DNA Replication Checkpoint Response in Fission Yeas

2007 ◽  
Vol 18 (5) ◽  
pp. 1756-1767 ◽  
Author(s):  
Zhaoqing Chu ◽  
Juntao Li ◽  
Majid Eshaghi ◽  
Xu Peng ◽  
R. Krishna M. Karuturi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
S Phase ◽  
Expression Level ◽  
Specific Gene ◽  
Dependent Manner ◽  
Replication Checkpoint ◽  
Checkpoint Kinases ◽  
Phase Arrest ◽  
S Phase Arrest

Fission yeast replication checkpoint kinases Rad3p and Cds1p are essential for maintaining cell viability after transient treatment with hydroxyurea (HU), an agent that blocks DNA replication. Although current studies have focused on the cyclin-dependent protein kinase Cdc2p that is regulated by these checkpoint kinases, other aspects of their functions at the onset of S phase arrest have not been fully understood. In this study, we use genome-wide DNA microarray analyses to show that HU-induced change of expression profiles in synchronized G2 cells occurs specifically at the onset of S phase arrest. Induction of many core environmental stress response genes and repression of ribosomal genes happen during S phase arrest. Significantly, peak expression level of the MluI-like cell cycle box (MCB)-cluster (G1) genes is maintained at the onset of S phase arrest in a Rad3p- and Cds1p-dependent manner. Expression level maintenance of the MCB-cluster is mediated through the accumulation of Rep2p, a putative transcriptional activator of the MBF complex. Conversely, the FKH-cluster (M) genes are repressed during the onset of S phase arrest in a Rad3p-dependent manner. Repression of the FKH-cluster genes is mediated through the decreased levels of one of the putative forkhead transcription factors, Sep1p, but not Fkh2p. Together, our results demonstrate that Rad3p and Cds1p modulate transcriptional response during the onset of S phase arrest.

scholarly journals Functions of Fission Yeast Orp2 in DNA Replication and Checkpoint Control

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 599-607
Author(s):  
Joan Kiely ◽  
S B Haase ◽  
Paul Russell ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Control ◽  
Cycle Arrest

Abstract orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.

scholarly journals DNA Replication Checkpoint Control Mediated by the Spindle Checkpoint Protein Mad2p in Fission Yeast

2004 ◽  
Vol 279 (45) ◽  
pp. 47372-47378 ◽  
Author(s):  
Izumi Sugimoto ◽  
Hiroshi Murakami ◽  
Yuko Tonami ◽  
Akihiko Moriyama ◽  
Makoto Nakanishi
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Spindle Checkpoint ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Checkpoint Protein ◽  
Dna Replication Checkpoint

Cdc18p can block mitosis by two independent mechanisms

1998 ◽  
Vol 111 (20) ◽  
pp. 3101-3108 ◽  
Author(s):  
E. Greenwood ◽  
H. Nishitani ◽  
P. Nurse
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Mitotic Kinase ◽  
C Terminus ◽  
N Terminus ◽  
Dna Replication Checkpoint ◽  
Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

scholarly journals Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

2001 ◽  
Vol 12 (5) ◽  
pp. 1257-1274 ◽  
Author(s):  
Tadayuki Takeda ◽  
Keiko Ogino ◽  
Kazuo Tatebayashi ◽  
Hideo Ikeda ◽  
Ken-ichi Arai ◽  
...  
Keyword(s):  
Dna Replication ◽  
Synthetic Lethality ◽  
S Phase ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Origin Firing ◽  
Replication Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Pathway ◽  
Nuclear Structures

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect inhsk1-89 is indicated by accumulation ofcut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling.hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those ofrad21-K1 and are significantly enhanced in ahsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

scholarly journals Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint

2008 ◽  
Vol 19 (2) ◽  
pp. 445-456 ◽  
Author(s):  
Benoît Froget ◽  
Joël Blaisonneau ◽  
Sarah Lambert ◽  
Giuseppe Baldacci
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Nuclease Activity ◽  
S Phase ◽  
Dna Breaks ◽  
Replication Checkpoint ◽  
Dna Structures ◽  
Replication Arrest ◽  
Dna Replication Checkpoint ◽  
Stalled Fork

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase–specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

scholarly journals Analysis of Fission Yeast Primase Defines the Checkpoint Responses to Aberrant S Phase Initiation

2000 ◽  
Vol 20 (21) ◽  
pp. 7853-7866 ◽  
Author(s):  
Siyuan Tan ◽  
Teresa S.-F. Wang
Keyword(s):  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Restrictive Temperature ◽  
Checkpoint Response ◽  
Dna Primase ◽  
Phase Arrest ◽  
S Phase Arrest ◽  
M Phase ◽  
Primase Complex ◽  
S Phase Checkpoint

ABSTRACT To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations ofSchizosaccharomyces 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 α (Polα) to synthesize an initiation DNA structure, and this catalytic function of Polα is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Polα. Thermosensitive mutations of spp2 +destabilize the Polα-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 Polα-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 polαtsmutant with a defective S-M phase checkpoint and anspp2 mutant with an intact checkpoint have a similar Polα-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 Polα.

The Spd1p S phase inhibitor can activate the DNA replication checkpoint pathway in fission yeast

2000 ◽  
Vol 113 (23) ◽  
pp. 4341-4350 ◽  
Author(s):  
A. Borgne ◽  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Checkpoint Control ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
A Cell ◽  
Checkpoint Genes

Spd1p (for S phase delayed) is a cell cycle inhibitor in Schizosaccharomyces pombe. Spd1p overexpression blocks the onset of both S phase and mitosis. In this study, we have investigated the mechanisms by which Spd1p overexpression blocks cell cycle progression, focussing on the block over mitotic onset. High levels of Spd1p lead to an increase in Y15 phosphorylation of Cdc2p and we show that the block over G(2) requires the Wee1p kinase and is dependent on the rad and chk1/cds1 checkpoint genes. We propose that high levels of Spd1p in G(2) cells activate the DNA replication checkpoint control, which leads to a Wee1p-dependent increase of Cdc2p Y15 phosphorylation blocking onset of mitosis. The Spd1p block at S phase onset may act by interfering directly with DNA replication, and also activates the G(2)rad/hus checkpoint pathway to block mitosis.

The fission yeast cdc19+ gene encodes a member of the MCM family of replication proteins

1994 ◽  
Vol 107 (10) ◽  
pp. 2779-2788 ◽  
Author(s):  
S.L. Forsburg ◽  
P. Nurse
Keyword(s):  
Fission Yeast ◽  
Dna Content ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Checkpoint Control ◽  
2C Dna Content ◽  
Synthetically Lethal ◽  
Structural Homologue ◽  
Mcm2 Protein

We have cloned and characterized the fission yeast cdc19+ gene. We demonstrate that it encodes a structural homologue of the budding yeast MCM2 protein. In fission yeast, the cdc19+ gene is constitutively expressed, and essential for viability. Deletion delays progression through S phase, and cells arrest in the first cycle with an apparent 2C DNA content, with their checkpoint control intact. The temperature-sensitive cdc19-P1 mutation is synthetically lethal with cdc21-M68. In addition, we show by classical and molecular genetics that cdc19+ is allelic to the nda1+ locus. We conclude that cdc19p plays a potentially conserved role in S phase.

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