A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

1980 ◽  
Vol 6 (4) ◽  
pp. 465-476 ◽  
Author(s):  
Takeharu Nishimoto ◽  
Taijo Takahashi ◽  
Claudio Basilico
Keyword(s):  
Dna Content ◽  
S Phase ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutation ◽  
Phase Progression

scholarly journals Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

2012 ◽  
Vol 23 (21) ◽  
pp. 4203-4211 ◽  
Author(s):  
Dong-Hwan Kim ◽  
Deanna M. Koepp
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Charged Residues ◽  
Phase Progression ◽  
Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

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 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.

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 Mutations in SID2, a Novel Gene in Saccharomyces cerevisiae, Cause Synthetic Lethality With sic1 Deletion and May Cause a Defect During S Phase

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 17-33
Author(s):  
Matthew D Jacobson ◽  
Claudia X Muñoz ◽  
Kirstin S Knox ◽  
Beth E Williams ◽  
Lenette L Lu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Synthetic Lethality ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Forks ◽  
Novel Gene ◽  
Mutant Strains ◽  
Single Nucleus ◽  
2C Dna Content ◽  
Slow Growing

Abstract SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis. To understand more completely the regulation of these transitions, mutations causing synthetic lethality with sic1Δ were isolated. In this screen, we identified a novel gene, SID2, which encodes an essential protein that appears to be required for DNA replication or repair. sid2-1 sic1Δ strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an ~2C DNA content. RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1Δ cells. Analysis of chromosomes in mutant sid2-21 cells by field inversion gel electrophoresis suggests the presence of replication forks and bubbles at the arrest. Deleting the two S phase cyclins, CLB5 and CLB6, substantially suppresses the sid2-1 sic1Δ inviability, while stabilizing Clb5 protein exacerbates the defects of sid2-1 sic1Δ cells. In synchronized sid2-1 mutant strains, the onset of replication appears normal, but completion of DNA synthesis is delayed. sid2-1 mutants are sensitive to hydroxyurea indicating that sid2-1 cells may suffer DNA damage that, when combined with additional insult, leads to a decrease in viability. Consistent with this hypothesis, sid2-1 rad9 cells are dead or very slow growing even when SIC1 is expressed.

scholarly journals The Meiosis-Specific Crs1 Cyclin Is Required for Efficient S-Phase Progression and Stable Nuclear Architecture

2021 ◽  
Vol 22 (11) ◽  
pp. 5483
Author(s):  
Luisa F. Bustamante-Jaramillo ◽  
Celia Ramos ◽  
Cristina Martín-Castellanos
Keyword(s):  
Cell Cycle ◽  
Translational Control ◽  
Cell Cycle Progression ◽  
Nuclear Architecture ◽  
Strand Break ◽  
Spindle Pole ◽  
S Phase ◽  
Cycle Progression ◽  
Single Wave ◽  
Phase Progression

Cyclins and CDKs (Cyclin Dependent Kinases) are key players in the biology of eukaryotic cells, representing hubs for the orchestration of physiological conditions with cell cycle progression. Furthermore, as in the case of meiosis, cyclins and CDKs have acquired novel functions unrelated to this primal role in driving the division cycle. Meiosis is a specialized developmental program that ensures proper propagation of the genetic information to the next generation by the production of gametes with accurate chromosome content, and meiosis-specific cyclins are widespread in evolution. We have explored the diversification of CDK functions studying the meiosis-specific Crs1 cyclin in fission yeast. In addition to the reported role in DSB (Double Strand Break) formation, this cyclin is required for meiotic S-phase progression, a canonical role, and to maintain the architecture of the meiotic chromosomes. Crs1 localizes at the SPB (Spindle Pole Body) and is required to stabilize the cluster of telomeres at this location (bouquet configuration), as well as for normal SPB motion. In addition, Crs1 exhibits CDK(Cdc2)-dependent kinase activity in a biphasic manner during meiosis, in contrast to a single wave of protein expression, suggesting a post-translational control of its activity. Thus, Crs1 displays multiple functions, acting both in cell cycle progression and in several key meiosis-specific events.

scholarly journals Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
Author(s):  
Xuan Wang ◽  
Grzegorz Ira ◽  
José Antonio Tercero ◽  
Allyson M. Holmes ◽  
John F. X. Diffley ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Dna Ligases ◽  
Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

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