scholarly journals Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2723-2734 ◽  
Author(s):  
X Li ◽  
M Cai
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
General Mechanism ◽  
Mitotic Spindles ◽  
Cycle Arrest

Eukaryotic cells may halt cell cycle progression following exposure to certain exogenous agents that damage cellular structures such as DNA or microtubules. This phenomenon has been attributed to functions of cellular control mechanisms termed checkpoints. Studies with the fission yeast Schizosaccharomyces pombe and mammalian cells have led to the conclusion that cell cycle arrest in response to inhibition of DNA replication or DNA damage is a result of down-regulation of the cyclin-dependent kinases (CDKs). Based on these studies, it has been proposed that inhibition of the CDK activity may constitute a general mechanism for checkpoint controls. Observations made with the budding yeast Saccharomyces cerevisiae, however, appear to disagree with this model. It has been shown that high levels of mitotic CDK activity are present in the budding yeast cells arrested in G2/mitosis as the result of DNA damage or replication inhibition. In this report, we show that a novel mutant allele of the CDC28 gene, encoding the budding yeast CDK, allowed cell cycle passage through mitosis and nuclear division in the presence of DNA damage and the microtubule toxin nocodazole at a restrictive temperature. Unlike the checkpoint-defective mutations in CDKs of fission yeast and mammalian cells, the cdc28 mutation that we identified was recessive and resulted in a loss of the CDK activity, including the Clb2-, Clb5-, and Clb6-associated, but not the Clb3-associated, CDK activities. Examination of several known alleles of cdc28 revealed that they were also, albeit partially, defective in cell cycle arrest in response to UV-generated DNA damage. These findings suggest that Cdc28 kinase in budding yeast may be required for cell cycle arrest resulting from DNA damage and disassembly of mitotic spindles.

scholarly journals Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

2009 ◽  
Vol 284 (52) ◽  
pp. 36191-36201 ◽  
Author(s):  
Christopher A. Koczor ◽  
Inna N. Shokolenko ◽  
Amy K. Boyd ◽  
Shawn P. Balk ◽  
Glenn L. Wilson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Mitochondrial Dna ◽  
Cell Cycle Arrest ◽  
Mammalian Cells ◽  
Mitochondrial Dna Damage ◽  
Cycle Arrest ◽  
A Cell

Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage

1990 ◽  
Vol 10 (12) ◽  
pp. 6554-6564
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Dna Sequence ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Protein Synthesis Inhibitor ◽  
Cycle Arrest ◽  
Delta Cells

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

scholarly journals Functional Complementation of Human Centromere Protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae

2004 ◽  
Vol 24 (15) ◽  
pp. 6620-6630 ◽  
Author(s):  
Gerhard Wieland ◽  
Sandra Orthaus ◽  
Sabine Ohndorf ◽  
Stephan Diekmann ◽  
Peter Hemmerich
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Budding Yeast ◽  
Protein A ◽  
Human Cells ◽  
Cycle Arrest ◽  
Centromere Protein ◽  
Centromere Protein A

ABSTRACT We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans.

scholarly journals A RAD9-dependent cell cycle arrest in response to unresolved recombination intermediates in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Hardeep Kaur ◽  
GN Krishnaprasad ◽  
Michael Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Cell Cycle Arrest ◽  
Mitotic Cell ◽  
Cycle Arrest ◽  
Associated Lesions

AbstractIn Saccharomyces cerevisiae, the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are precursors of crossovers. In vitro studies have suggested that the dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation could be responsible for this. To ask if dissolution occurs in vivo, we conditionally depleted Sgs1 and/or Rmi1 during return to growth, a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle. Sgs1 depletion during return to growth delayed joint molecule resolution, but ultimately most were resolved and cells divided normally. In contrast, Rmi1 depletion resulted in delayed and incomplete joint molecule resolution, and most cells did not divide. rad9Δ mutation restored cell division in Rmi1-depleted cells, indicating that the DNA damage checkpoint caused this cell cycle arrest. Restored cell division in rad9Δ, Rmi1-depleted cells frequently produced anucleate cells, consistent with the suggestion that persistent recombination intermediates prevented chromosome segregation. Our findings indicate that Sgs1-Top3-Rmi1 acts in vivo, as it does in vitro, to promote recombination intermediate resolution by dissolution. They also indicate that, in the absence of Top3-Rmi1 activity, unresolved recombination intermediates persist and activate the DNA damage response, which is usually thought to be activated by much earlier DNA damage-associated lesions.

scholarly journals Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage.

1989 ◽  
Vol 9 (5) ◽  
pp. 1882-1896 ◽  
Author(s):  
R H Schiestl ◽  
P Reynolds ◽  
S Prakash ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Chain Elongation ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dna Breaks ◽  
Cycle Arrest ◽  
Delta Cells

Procaryotic and eucaryotic cells possess mechanisms for arresting cell division in response to DNA damage. Eucaryotic cells arrest division in the G2 stage of the cell cycle, and various observations suggest that this arrest is necessary to ensure the completion of repair of damaged DNA before the entry of cells into mitosis. Here, we provide evidence that the Saccharomyces cerevisiae RAD9 gene, mutations of which confer sensitivity to DNA-damaging agents, is necessary for the cell cycle arrest phenomenon. Our studies with the rad9 delta mutation show that RAD9 plays a role in the cell cycle arrest of methyl methanesulfonate-treated cells and is absolutely required for the cell cycle arrest in the temperature-sensitive cdc9 mutant, which is defective in DNA ligase. At the restrictive temperature, cell cycle progression of cdc9 cells is blocked sometime after the DNA chain elongation step, whereas cdc9 rad9 delta cells do not arrest at this point and undergo one or two additional divisions. Upon transfer from the restrictive to the permissive temperature, a larger proportion of the cdc9 cells than of the cdc9 rad9 delta cells forms viable colonies, indicating that RAD9-mediated cell cycle arrest allows for proper ligation of DNA breaks before the entry of cells into mitosis. The rad9 delta mutation does not affect the frequency of spontaneous or UV-induced mutation and recombination, suggesting that RAD9 is not directly involved in mutagenic or recombinational repair processes. The RAD9 gene encodes a transcript of approximately 4.2 kilobases and a protein of 1,309 amino acids of Mr 148,412. We suggest that RAD9 may be involved in regulating the expression of genes required for the transition from G2 to mitosis.

scholarly journals Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.

1990 ◽  
Vol 10 (12) ◽  
pp. 6554-6564 ◽  
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Dna Sequence ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Protein Synthesis Inhibitor ◽  
Cycle Arrest ◽  
Delta Cells

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

scholarly journals Modelling the checkpoint response to telomere uncapping in budding yeast

2006 ◽  
Vol 4 (12) ◽  
pp. 73-90 ◽  
Author(s):  
C.J Proctor ◽  
D.A Lydall ◽  
R.J Boys ◽  
C.S Gillespie ◽  
D.P Shanley ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Dna Damage Response ◽  
Time Course ◽  
Budding Yeast ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Damage Response

One of the DNA damage-response mechanisms in budding yeast is temporary cell-cycle arrest while DNA repair takes place. The DNA damage response requires the coordinated interaction between DNA repair and checkpoint pathways. Telomeres of budding yeast are capped by the Cdc13 complex. In the temperature-sensitive cdc13-1 strain, telomeres are unprotected over a specific temperature range leading to activation of the DNA damage response and subsequently cell-cycle arrest. Inactivation of cdc13-1 results in the generation of long regions of single-stranded DNA (ssDNA) and is affected by the activity of various checkpoint proteins and nucleases. This paper describes a mathematical model of how uncapped telomeres in budding yeast initiate the checkpoint pathway leading to cell-cycle arrest. The model was encoded in the Systems Biology Markup Language (SBML) and simulated using the stochastic simulation system Biology of Ageing e-Science Integration and Simulation (BASIS). Each simulation follows the time course of one mother cell keeping track of the number of cell divisions, the level of activity of each of the checkpoint proteins, the activity of nucleases and the amount of ssDNA generated. The model can be used to carry out a variety of in silico experiments in which different genes are knocked out and the results of simulation are compared to experimental data. Possible extensions to the model are also discussed.

Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage

1989 ◽  
Vol 9 (5) ◽  
pp. 1882-1896
Author(s):  
R H Schiestl ◽  
P Reynolds ◽  
S Prakash ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Chain Elongation ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dna Breaks ◽  
Cycle Arrest ◽  
Delta Cells

Procaryotic and eucaryotic cells possess mechanisms for arresting cell division in response to DNA damage. Eucaryotic cells arrest division in the G2 stage of the cell cycle, and various observations suggest that this arrest is necessary to ensure the completion of repair of damaged DNA before the entry of cells into mitosis. Here, we provide evidence that the Saccharomyces cerevisiae RAD9 gene, mutations of which confer sensitivity to DNA-damaging agents, is necessary for the cell cycle arrest phenomenon. Our studies with the rad9 delta mutation show that RAD9 plays a role in the cell cycle arrest of methyl methanesulfonate-treated cells and is absolutely required for the cell cycle arrest in the temperature-sensitive cdc9 mutant, which is defective in DNA ligase. At the restrictive temperature, cell cycle progression of cdc9 cells is blocked sometime after the DNA chain elongation step, whereas cdc9 rad9 delta cells do not arrest at this point and undergo one or two additional divisions. Upon transfer from the restrictive to the permissive temperature, a larger proportion of the cdc9 cells than of the cdc9 rad9 delta cells forms viable colonies, indicating that RAD9-mediated cell cycle arrest allows for proper ligation of DNA breaks before the entry of cells into mitosis. The rad9 delta mutation does not affect the frequency of spontaneous or UV-induced mutation and recombination, suggesting that RAD9 is not directly involved in mutagenic or recombinational repair processes. The RAD9 gene encodes a transcript of approximately 4.2 kilobases and a protein of 1,309 amino acids of Mr 148,412. We suggest that RAD9 may be involved in regulating the expression of genes required for the transition from G2 to mitosis.

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