scholarly journals Checkpoint regulation of nuclear Tos4 defines S phase arrest in fission yeast

2019 ◽  
Author(s):  
Seong M. Kim ◽  
Vishnu P. Tripathi ◽  
Kuo-Fang Shen ◽  
Susan L. Forsburg
Keyword(s):  
Dna Content ◽  
Regulatory Networks ◽  
S Phase ◽  
Nuclear Accumulation ◽  
Restrictive Temperature ◽  
Checkpoint Kinases ◽  
Phase Arrest ◽  
Visual Markers ◽  
2C Dna Content ◽  
Phase Progression

ABSTRACTFrom yeast to humans, the cell cycle is tightly controlled by regulatory networks that regulate cell proliferation and can be monitored by dynamic visual markers in living cells. We have observed S phase progression by monitoring nuclear accumulation of the FHA-containing DNA binding protein Tos4, which is expressed in the G1/S phase transition. We use Tos4 localization to distinguish three classes of DNA replication mutants: those that arrest with an apparent 1C DNA content and accumulate Tos4 at the restrictive temperature; those that arrest with an apparent 2C DNA content, that do not accumulate Tos4; and those that proceed into mitosis despite a 1C DNA content, again without Tos4 accumulation. Our data indicate that Tos4 localization in these conditions is responsive to checkpoint kinases, with activation of the Cds1 checkpoint kinase promoting Tos4 retention in the nucleus, and activation of the Chk1 damage checkpoint promoting its turnover. Tos4 localization therefore allows us to monitor checkpoint-dependent activation that responds to replication failure in early versus late S phase.

scholarly journals Checkpoint Regulation of Nuclear Tos4 Defines S Phase Arrest in Fission Yeast

2019 ◽  
Vol 10 (1) ◽  
pp. 255-266 ◽  
Author(s):  
Seong M. Kim ◽  
Vishnu P. Tripathi ◽  
Kuo-Fang Shen ◽  
Susan L. Forsburg
Keyword(s):  
Dna Content ◽  
Regulatory Networks ◽  
S Phase ◽  
Nuclear Accumulation ◽  
Restrictive Temperature ◽  
Checkpoint Kinases ◽  
Phase Arrest ◽  
Visual Markers ◽  
2C Dna Content ◽  
Phase Progression

From yeast to humans, the cell cycle is tightly controlled by regulatory networks that regulate cell proliferation and can be monitored by dynamic visual markers in living cells. We have observed S phase progression by monitoring nuclear accumulation of the FHA-containing DNA binding protein Tos4, which is expressed in the G1/S phase transition. We use Tos4 localization to distinguish three classes of DNA replication mutants: those that arrest with an apparent 1C DNA content and accumulate Tos4 at the restrictive temperature; those that arrest with an apparent 2C DNA content, that do not accumulate Tos4; and those that proceed into mitosis despite a 1C DNA content, again without Tos4 accumulation. Our data indicate that Tos4 localization in these conditions is responsive to checkpoint kinases, with activation of the Cds1 checkpoint kinase promoting Tos4 retention in the nucleus, and activation of the Chk1 damage checkpoint promoting its turnover. Tos4 localization therefore allows us to monitor checkpoint-dependent activation that responds to replication failure in early vs. late S phase.

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 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 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.

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 The Role of Nucleotide Binding and Hydrolysis in the Function of the Fission Yeast cdc18+ Gene Product

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1445-1457
Author(s):  
Deborah DeRyckere ◽  
Cheryl L Smith ◽  
G Steven Martin
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Gene Product ◽  
Dna Content ◽  
Nucleotide Binding ◽  
Mitotic Checkpoint ◽  
S Phase ◽  
Wild Type ◽  
Mutant Strains ◽  
2C Dna Content

Abstract The fission yeast cdc18+ gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18+ gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C–2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.

scholarly journals Nuclear Accumulation of the Papillomavirus E1 Helicase Blocks S-Phase Progression and Triggers an ATM-Dependent DNA Damage Response

2011 ◽  
Vol 85 (17) ◽  
pp. 8996-9012 ◽  
Author(s):  
A. Fradet-Turcotte ◽  
F. Bergeron-Labrecque ◽  
C. A. Moody ◽  
M. Lehoux ◽  
L. A. Laimins ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
S Phase ◽  
Nuclear Accumulation ◽  
Damage Response ◽  
Phase Progression

scholarly journals Evidence That the pre-mRNA Splicing Factor Clf1p Plays a Role in DNA Replication in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1319-1333
Author(s):  
Wenge Zhu ◽  
Irene R Rainville ◽  
Min Ding ◽  
Margaret Bolus ◽  
Nicholas H Heintz ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Replication Initiation ◽  
Mrna Splicing ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Direct Role ◽  
Dna Replication Initiation ◽  
Single Nucleus ◽  
2C Dna Content

Abstract Clf1p is an essential, highly conserved protein in S. cerevisiae that has been implicated in pre-mRNA splicing. Clf1p's ortholog in Drosophila, Crn, is required for normal cell proliferation. Cells depleted of Clf1p arrest primarily with large buds, a single nucleus, a 2C DNA content, and a short, intact mitotic spindle. We isolated temperature-sensitive clf1 mutants that exhibit similar mitotic defects when released to the restrictive temperature from an early S-phase block. While these mutants also accumulate unspliced pre-mRNA at the restrictive temperature, the mitotic arrest does not appear to result from a failure to splice tubulin pre-mRNA. Moreover, the same mutants exhibit a delayed entry into S phase when released to the restrictive temperature from a G1 phase block. This delay could not be suppressed by disruption of the S-phase CDK inhibitor SIC1, suggesting that Clf1p is involved in DNA replication. Consistent with this possibility, we find that Clf1p (but not the mutant clf1p) interacts with the DNA replication initiation protein Orc2p in two-hybrid and co-immunoprecipitation assays, that Clf1p preferentially associates with origins of DNA replication, and that this association is Orc2p dependent. These observations suggest that Clf1p plays a direct role in the initiation of DNA replication.

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