scholarly journals Involvement of the PP2C-Like Phosphatase Ptc2p in the DNA Checkpoint Pathways of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1523-1532
Author(s):  
Marie-Claude Marsolier ◽  
Pascal Roussel ◽  
Christophe Leroy ◽  
Carl Mann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Protein Kinase ◽  
Ribonucleotide Reductase ◽  
Protein Phosphatase ◽  
Reductase Activity ◽  
Wild Type ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint ◽  
Ribonucleotide Reductase Activity

Abstract RAD53 encodes a conserved protein kinase that acts as a central transducer in the DNA damage and the DNA replication checkpoint pathways in Saccharomyces cerevisiae. To identify new elements of these pathways acting with or downstream of RAD53, we searched for genes whose overexpression suppressed the toxicity of a dominant-lethal form of RAD53 and identified PTC2, which encodes a protein phosphatase of the PP2C family. PTC2 overexpression induces hypersensitivity to genotoxic agents in wild-type cells and is lethal to rad53, mec1, and dun1 mutants with low ribonucleotide reductase activity. Deleting PTC2 specifically suppresses the hydroxyurea hypersensitivity of mec1 mutants and the lethality of mec1Δ. PTC2 is thus implicated in one or several functions related to RAD53, MEC1, and the DNA checkpoint pathways.

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells

1983 ◽  
Vol 3 (8) ◽  
pp. 741-748 ◽  
Author(s):  
Jim A. Wright ◽  
Joseph G. Cory
Keyword(s):  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Antitumor Agent ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Wild Type ◽  
Type Analysis ◽  
Similar Mode ◽  
Ribonucleotide Reductase Activity ◽  
Effector Binding

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NcR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NcR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heine-ironcontaining (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NcR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.

Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1081-1087 ◽  
Author(s):  
Robert A. R. Hurta ◽  
Jim A. Wright
Keyword(s):  
Okadaic Acid ◽  
Ribonucleotide Reductase ◽  
Protein Phosphatase ◽  
Reductase Activity ◽  
Tumor Promoter ◽  
Calyculin A ◽  
Tumor Promoters ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Ribonucleotide Reductase Activity

A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment. This first demonstration that the non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause rapid alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, particularly the R2 component, plays a fundamental role in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis.Key words: ribonucleotide reductase, DNA synthesis, okadaic acid, calyculin A, tumor promoter, protein phosphatase.

scholarly journals Increased Genome Instability and Telomere Length in the elg1-Deficient Saccharomyces cerevisiae Mutant Are Regulated by S-Phase Checkpoints

2004 ◽  
Vol 3 (6) ◽  
pp. 1557-1566 ◽  
Author(s):  
Soma Banerjee ◽  
Kyungjae Myung
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Telomere Length ◽  
Genome Instability ◽  
Dna Recombination ◽  
Cell Cycle Checkpoints ◽  
Telomere Maintenance ◽  
Replication Checkpoint ◽  
Dna Replication Checkpoint

ABSTRACT Gross chromosomal rearrangements (GCRs) are frequently observed in cancer cells. Abnormalities in different DNA metabolism including DNA replication, cell cycle checkpoints, chromatin remodeling, telomere maintenance, and DNA recombination and repair cause GCRs in Saccharomyces cerevisiae. Recently, we used genome-wide screening to identify several genes the deletion of which increases GCRs in S. cerevisiae. Elg1, which was discovered during this screening, functions in DNA replication by participating in an alternative replication factor complex. Here we further characterize the GCR suppression mechanisms observed in the elg1Δ mutant strain in conjunction with the telomere maintenance role of Elg1. The elg1Δ mutation enhanced spontaneous DNA damage and resulted in GCR formation. However, DNA damage due to inactivation of Elg1 activates the intra-S checkpoints, which suppress further GCR formation. The intra-S checkpoints activated by the elg1Δ mutation also suppress GCR formation in strains defective in the DNA replication checkpoint. Lastly, the elg1Δ mutation increases telomere size independently of other previously known telomere maintenance proteins such as the telomerase inhibitor Pif1 or the telomere size regulator Rif1. The increase in telomere length caused by the elg1Δ mutation was suppressed by a defect in the DNA replication checkpoint, which suggests that DNA replication surveillance by Dpb11-Mec1/Tel1-Dun1 also has an important role in telomere length regulation.

scholarly journals Loss of SOD1 and LYS7 Sensitizes Saccharomyces cerevisiae to Hydroxyurea and DNA Damage Agents and Downregulates MEC1 Pathway Effectors

2005 ◽  
Vol 25 (23) ◽  
pp. 10273-10285 ◽  
Author(s):  
Carole D. Carter ◽  
Lauren E. Kitchen ◽  
Wei-Chun Au ◽  
Christopher M. Babic ◽  
Munira A. Basrai
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Reductase Activity ◽  
Copper Chaperone ◽  
Superoxide Anions ◽  
Cellular Redox ◽  
Checkpoint Response ◽  
Replication Arrest ◽  
Mutant Strains ◽  
Ribonucleotide Reductase Activity

ABSTRACT Aerobic metabolism produces reactive oxygen species, including superoxide anions, which cause DNA damage unless removed by scavengers such as superoxide dismutases. We show that loss of the Cu,Zn-dependent superoxide dismutase, SOD1, or its copper chaperone, LYS7, confers oxygen-dependent sensitivity to replication arrest and DNA damage in Saccharomyces cerevisiae. We also find that sod1Δ strains, and to a lesser extent lys7Δ strains, when arrested with hydroxyurea (HU) show reduced induction of the MEC1 pathway effector Rnr3p and of Hug1p. The HU sensitivity of sod1Δ and lys7Δ strains is suppressed by overexpression of TKL1, a transketolase that generates NADPH, which balances redox in the cell and is required for ribonucleotide reductase activity. Our results suggest that the MEC1 pathway in sod1Δ mutant strains is sensitive to the altered cellular redox state due to increased superoxide anions and establish a new relationship between SOD1, LYS7, and the MEC1-mediated checkpoint response to replication arrest and DNA damage in S. cerevisiae.

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines

1991 ◽  
Vol 69 (9) ◽  
pp. 635-642 ◽  
Author(s):  
Robert A. R. Hurta ◽  
Jim A. Wright
Keyword(s):  
Enzyme Activity ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Protein Concentration ◽  
Reductase Activity ◽  
M2 Protein ◽  
Wild Type ◽  
L Cells ◽  
Ribonucleotide Reductase Activity

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity. This is the first demonstration that reversion of hydroxyurea resistance and a decline in ribonucleotide reductase activity are accompanied by decreased ferritin expression, and supports the concept that ferritin is important in establishing resistance to hydroxyurea, and may play a role in DNA synthesis, through the regulation of functional iron-containing M2 protein levels required for ribonucleotide reduction.Key words: ribonucleotide reductase, ferritin, hydroxyurea, drug resistance.

scholarly journals Cellular adaptation to down-regulated iron transport into lymphoid leukaemic cells: effects on the expression of the gene for ribonucleotide reductase

2000 ◽  
Vol 345 (3) ◽  
pp. 681-685 ◽  
Author(s):  
Christopher R. CHITAMBAR ◽  
Janine P. WERELEY ◽  
Thomas HEIMAN ◽  
William E. ANTHOLINE ◽  
William J. O'BRIEN
Keyword(s):  
T Cells ◽  
Enzyme Activity ◽  
Ribonucleotide Reductase ◽  
Iron Transport ◽  
Reductase Activity ◽  
Wild Type ◽  
Cellular Iron ◽  
Regulatory Link ◽  
The Relationship ◽  
Ribonucleotide Reductase Activity

Ribonucleotide reductase is an iron-containing enzyme that is essential for DNA synthesis. Whereas previous studies have used various iron chelators to examine the relationship between cellular iron metabolism and ribonucleotide reductase activity in cells, they have not elucidated the relationship between iron transport into cells and the expression of the gene for ribonucleotide reductase. To investigate this, we examined ribonucleotide reductase mRNA, protein and enzyme activity in a novel line of CCRF-CEM cells (DFe-T cells) that display an approx. 60% decrease in their uptake of iron compared with the parental wild-type cell line. We found that DFe-T cells displayed an approx. 40% decrease in ribonucleotide reductase specific enzyme activity relative to wild-type cells without a change in their proliferation. Kinetic analysis of CDP reductase activity revealed an approx. 60% decrease in Vmax in DFe-T cells without a change in Km. Despite the decrease in enzyme activity, the mRNA and protein for the R1 and R2 subunits of ribonucleotide reductase in DFe-T cells were similar to those of wild-type cells. ESR spectroscopy studies revealed that DFe-T cells had a 22% decrease in the tyrosyl free radical of the R2 subunit, suggesting that a larger amount of R2 protein was present as functionally inactive apo-R2 in these cells. Our studies indicate that ribonucleotide reductase activity in CCRF-CEM cells can be down-regulated by more than 50% in response to down-regulated iron transport without an adverse effect on cell proliferation. Furthermore, our studies suggest a regulatory link between ribonucleotide reductase activity and iron transport into these cells.

Overlapping Roles of the Spindle Assembly and DNA Damage Checkpoints in the Cell-Cycle Response to Altered Chromosomes in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 521-534
Author(s):  
Peter M Garber ◽  
Jasper Rine
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Spindle Checkpoint ◽  
Dna Lesions ◽  
Replication Proteins ◽  
Replication Checkpoint ◽  
Dna Damage Checkpoints ◽  
Dna Replication Checkpoint ◽  
Mcm Proteins ◽  
Stalled Replication Forks

Abstract The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Δ mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase δ. Thus the specificity of this checkpoint may be more limited than previously recognized.

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