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

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6554-6564.1990 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6554 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6554-6564 ◽

Cited By ~ 121

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.

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

10.1101/736850 ◽

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.

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HIV Vpr Modulates the Host DNA Damage Response at Two Independent Steps to Damage DNA and Repress Double-Strand DNA Break Repair

mBio ◽

10.1128/mbio.00940-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Donna Li ◽

Andrew Lopez ◽

Carina Sandoval ◽

Randilea Nichols Doyle ◽

Oliver I. Fregoso

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Arrest ◽

Dna Damage Response ◽

Signaling Cascade ◽

Human Pathogens ◽

Dna Breaks ◽

Cycle Arrest ◽

Damage Response ◽

Hiv 1

ABSTRACT The DNA damage response (DDR) is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. Previous work has shown that HIV-1 is also capable of engaging the host DDR, primarily through the conserved accessory protein Vpr. However, the extent of this engagement has remained unclear. Here, we show that HIV-1 and HIV-2 Vpr directly induce DNA damage and stall DNA replication, leading to the activation of several markers of double- and single-strand DNA breaks. Despite causing damage and activating the DDR, we found that Vpr represses the repair of double-strand breaks (DSB) by inhibiting homologous recombination (HR) and nonhomologous end joining (NHEJ). Mutational analyses of Vpr revealed that DNA damage and DDR activation are independent from repression of HR and Vpr-mediated cell cycle arrest. Moreover, we show that repression of HR does not require cell cycle arrest but instead may precede this long-standing enigmatic Vpr phenotype. Together, our data uncover that Vpr globally modulates the host DDR at at least two independent steps, offering novel insight into the primary functions of lentiviral Vpr and the roles of the DNA damage response in lentiviral replication. IMPORTANCE The DNA damage response (DDR) is a signaling cascade that safeguards the genome from genotoxic agents, including human pathogens. However, the DDR has also been utilized by many pathogens, such as human immunodeficiency virus (HIV), to enhance infection. To properly treat HIV-positive individuals, we must understand how the virus usurps our own cellular processes. Here, we have found that an important yet poorly understood gene in HIV, Vpr, targets the DDR at two unique steps: it causes damage and activates DDR signaling, and it represses the ability of cells to repair this damage, which we hypothesize is central to the primary function of Vpr. In clarifying these important functions of Vpr, our work highlights the multiple ways human pathogens engage the DDR and further suggests that modulation of the DDR is a novel way to help in the fight against HIV.

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HIV Vpr modulates the host DNA damage response at two independent steps to damage DNA and repress double-strand DNA break repair

10.1101/2020.04.26.062349 ◽

2020 ◽

Author(s):

Donna Li ◽

Andrew Lopez ◽

Carina Sandoval ◽

Randilea Nichols Doyle ◽

Oliver I Fregoso

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Arrest ◽

Dna Damage Response ◽

Signaling Cascade ◽

Human Pathogens ◽

Dna Breaks ◽

Cycle Arrest ◽

Damage Response ◽

Hiv 1

ABSTRACTThe DNA damage response (DDR) is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. Previous work has shown that HIV-1 is also capable of engaging the host DDR, primarily through the conserved accessory protein Vpr. However, the extent of this engagement has remained unclear. Here we show that HIV-1 and HIV-2 Vpr directly induce DNA damage and stall DNA replication, leading to the activation of several markers of double- and single-strand DNA breaks. Despite causing damage and activating the DDR, we found that Vpr repress the repair of double-strand breaks (DSB) by inhibiting homologous recombination (HR) and non-homologous end joining (NHEJ). Mutational analyses of Vpr revealed that DNA damage and DDR activation are independent from repression of HR and Vpr-mediated cell-cycle arrest. Moreover, we show that repression of HR does not require cell-cycle arrest but instead may precede this long-standing enigmatic Vpr phenotype. Together, our data uncover that Vpr globally modulates the host DDR at at least two independent steps; offering novel insight into the primary functions of lentiviral Vpr and the roles of the DNA damage response in lentiviral replication.IMPORTANCEThe DNA damage response (DDR) is a signaling cascade that safeguards the genome from genotoxic agents, including human pathogens. However, the DDR has also been utilized by many pathogens, such as Human Immunodeficiency Virus (HIV), to enhance infection. To properly treat HIV positive individuals, we must understand how the virus usurps our own cellular processes. Here, we have found that an important yet poorly-understood gene in HIV, Vpr, targets the DDR at two unique steps: it causes damage and activates DDR signaling, and it represses the ability of cells to repair this damage, which we hypothesize is central to the primary function of Vpr. In clarifying these important functions of Vpr, our work highlights the multiple ways human pathogens engage the DDR, and further suggests that modulation of the DDR may be a novel way to help in the fight against HIV.

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MYC Inactivation Induces Tumor Regression through the Recovery of a Functional DNA Damage Response.

Blood ◽

10.1182/blood.v104.11.1534.1534 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1534-1534

Author(s):

Karen Rabin ◽

Sylvie Giuriato ◽

Suma Ray ◽

Dean W. Felsher

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Arrest ◽

Tumor Cells ◽

Dna Damage Response ◽

Tumor Regression ◽

Dna Breaks ◽

Cycle Arrest ◽

Damage Response ◽

Functional Dna

Abstract Cancer is caused by genetic events that result in the activation of oncogenes or the inactivation of tumor suppressor genes. Using the Tetracycline system, our laboratory has generated a transgenic mouse model in which MYC is conditionally overexpressed in hematopoietic cells, allowing us to turn MYC expression on and off at will. We have previously demonstrated that the inactivation of this single oncogene in an established and even highly invasive and metastatic lymphoma is sufficient to reverse cancer, suggesting that MYC may be an effective therapeutic target. In a variety of conditional oncogene models, we and others have found that tumor regression following oncogene inactivation involves similar phenomena, including cell cycle arrest, apoptosis, and differentiation of the tumor cells. The similarity in the regression process across a variety of different oncogenes and types of cancer strongly suggests the existence of a common signaling pathway following oncogene inactivation, which culminates in the cessation of cell proliferation and the induction of apoptosis and differentiation. Recently, we have demonstrated that MYC activation disrupts the repair of DNA breaks and results in genomic instability. We speculated that MYC inactivation may cause tumor regression by restoring the ability of tumor cells to recognize that they are genomically damaged, which could subsequently lead to the cell cycle arrest, differentiation and apoptosis that is generally observed. Indeed, we now report that upon MYC inactivation, tumor cells activate DNA damage signaling pathways and begin to repair their DNA breaks. We have found evidence for activation of a functional DNA damage response both by immunofluorescent staining for phosphorylated ATM and Mre11, which demonstrates foci formation following MYC inactivation; and by the Comet assay, which shows a quantitative decrease in severity of DNA breaks following MYC inactivation. Our results suggest that MYC inactivation may induce tumor regression at least in part through the restoration of a DNA damage checkpoint response.

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2723 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2723-2734 ◽

Cited By ~ 32

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.

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Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans Induces DNA Damage, S/G2 Cell Cycle Arrest, and Caspase- Independent Death in a Saccharomyces cerevisiae Model

Infection and Immunity ◽

10.1128/iai.00857-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 783-792 ◽

Cited By ~ 21

Author(s):

Oranart Matangkasombut ◽

Roongtiwa Wattanawaraporn ◽

Keiko Tsuruda ◽

Masaru Ohara ◽

Motoyuki Sugai ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Cell Cycle Arrest ◽

Aggregatibacter Actinomycetemcomitans ◽

Cytolethal Distending Toxin ◽

Dependent Manner ◽

Cycle Arrest ◽

Yeast Strains

ABSTRACT Cytolethal distending toxin (CDT) is a bacterial toxin that induces G2/M cell cycle arrest, cell distension, and/or apoptosis in mammalian cells. It is produced by several Gram-negative species and may contribute to their pathogenicity. The catalytic subunit CdtB has homology with DNase I and may act as a genotoxin. However, the mechanism by which CdtB leads to cell death is not yet clearly understood. Here, we used Saccharomyces cerevisiae as a model to study the molecular pathways involved in the function of CdtB from Aggregatibacter actinomycetemcomitans, a cause of aggressive periodontitis. We show that A. actinomycetemcomitans CdtB (AaCdtB) expression induces S/G2 arrest and death in a DNase-catalytic residue and nuclear localization-dependent manner in haploid yeasts. Yeast strains defective in homologous recombination (HR) repair, but not other DNA repair pathways, are hypersensitive to AaCdtB, suggesting that HR is required for survival upon CdtB expression. In addition, yeast does not harbor the substrate for the other activity proposed for CdtB function, which is phosphatidylinositol-3,4,5-triphosphate phosphatase. Thus, these results suggest that direct DNA-damaging activity alone is sufficient for CdtB toxicity. To investigate how CdtB induces cell death, we examined the effect of CdtB in yeast strains with mutations in apoptotic regulators. Our results suggest that yeast death occurs independently of the yeast metacaspase gene YCA1 and the apoptosis-inducing factor AIF1 but is partially dependent on histone H2B serine 10 phosphorylation. Therefore, we report here the evidence that AaCdtB causes DNA damage that leads to nonapoptotic death in yeast and the first mutation that confers resistance to CdtB.

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