The origin recognition complex in silencing, cell cycle progression, and DNA replication.

This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes.

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The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression

Journal of Cell Science ◽

10.1242/jcs.113.7.1199 ◽

2000 ◽

Vol 113 (7) ◽

pp. 1199-1211

Author(s):

G. Buscemi ◽

F. Saracino ◽

D. Masnada ◽

M.L. Carbone

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Actin Cytoskeleton ◽

Cell Cycle Progression ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Cycle Progression ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.

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MEIOTIC RECOMBINATION AND DNA SYNTHESIS IN A NEW CELL CYCLE MUTANT OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/90.1.49 ◽

1978 ◽

Vol 90 (1) ◽

pp. 49-68

Author(s):

Yona Kassir ◽

Giora Simchen

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Dna Synthesis ◽

Nuclear Division ◽

Temperature Shift ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Essential Function ◽

Normal Meiosis

ABSTRACT Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

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Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4594-4601.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4594-4601

Author(s):

J J Dermody ◽

B E Wojcik ◽

H Du ◽

H L Ozer

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Chinese Hamster ◽

Human Adenovirus ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Dependent Manner ◽

Viral Dna ◽

Cell Functions

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

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Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽

10.1093/genetics/134.1.63 ◽

1993 ◽

Vol 134 (1) ◽

pp. 63-80 ◽

Cited By ~ 9

Author(s):

T A Weinert ◽

L H Hartwell

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Control ◽

Dna Lesions ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

A Cell ◽

G2 Phase ◽

Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

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Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5767-5777.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5767-5777 ◽

Cited By ~ 54

Author(s):

Amit Vas ◽

Winnie Mok ◽

Janet Leatherwood

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Origin Recognition Complex ◽

Safety Net ◽

Replication Initiation ◽

Initiation Factor ◽

Phosphorylation Sites ◽

Cdc2 Kinase ◽

Origin Recognition ◽

Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

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Mutations in RAD27 define a potential link between G1 cyclins and DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4291 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4291-4302 ◽

Cited By ~ 64

Author(s):

E A Vallen ◽

F R Cross

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Cell Cycle Progression ◽

Spontaneous Mutation ◽

Chromosome Loss ◽

Recombination Rates ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Increased Sensitivity

The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions. To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2. We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2. rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to a DNA-damaging agent, increased mitotic recombination rates, and increased spontaneous mutation rates. Unlike the block in cell cycle progression, these phenotypes are not suppressed by CLN1 or CLN2. CLN1 and CLN2 may activate an RAD27/ERC11-independent pathway specific for DNA synthesis that CLN3 is incapable of activating. Alternatively, CLN1 and CLN2 may be capable of overriding a checkpoint response which otherwise causes cln1 cln2 rad27/erc11 cells to arrest. These results imply that CLN1 and CLN2 have a role in the regulation of DNA replication. Consistent with this, GAL-CLN1 expression in checkpoint-deficient, mec1-1 mutant cells results in both cell death and increased chromosome loss among survivors, suggesting that CLN1 overexpression either activates defective DNA replication or leads to insensitivity to DNA damage.

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p53-mediated cell death: relationship to cell cycle control.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1415 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1415-1423 ◽

Cited By ~ 193

Author(s):

E Yonish-Rouach ◽

D Grunwald ◽

S Wilder ◽

A Kimchi ◽

E May ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Interleukin 6 ◽

Cell Cycle Progression ◽

P53 Protein ◽

Cell Cycle Control ◽

Growth Arrest ◽

Leukemic Cells ◽

Temperature Sensitive ◽

Permissive Temperature

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.

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The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae

10.1101/2021.05.14.444167 ◽

2021 ◽

Author(s):

Allegra Terhorst ◽

Arzu Sandikci ◽

Gabriel E. Neurohr ◽

Charles A. Whittaker ◽

Tamás Szórádi ◽

...

Keyword(s):

Cell Cycle ◽

Stress Response ◽

Environmental Stress ◽

Cell Cycle Progression ◽

Transcriptional Response ◽

Biomass Accumulation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Environmental Stress Response ◽

Pka Pathway

Temperature sensitive cell division cycle (cdc-ts) cells are unable to progress through the cell cycle at the restrictive temperature due to mutations in genes essential to cell cycle progress. Cells harboring cdc-ts mutations increase in cell volume upon arrest but eventually stop growing. We found that this attenuation in growth was due to selective downregulation of ribosome concentration. We saw similar ribosome downregulation in cells arrested in the cell cycle through alpha factor addition, rapamycin addition, and entrance into stationary phase. In all cell cycle arrests studied, cells activated the Environmental Stress Response (ESR), a key transcriptional response to many stressors in S. cerevisiae. When we combined cell cycle arrest with hyperactivation of the Ras/PKA pathway, ESR activation was prevented, cells were unable to downregulate their ribosomes, and cell viability was decreased. Our work uncovers a key role for the environmental stress response in coupling cell cycle progression to biomass accumulation.

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Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation

Journal of Cell Science ◽

10.1242/jcs.108.3.895 ◽

1995 ◽

Vol 108 (3) ◽

pp. 895-905 ◽

Cited By ~ 8

Author(s):

K. Kumada ◽

S. Su ◽

M. Yanagida ◽

T. Toda

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Fission Yeast ◽

Nitrogen Starvation ◽

G1 Arrest ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Septum Formation ◽

Family Protein ◽

Chromosome Separation

Fission yeast nuc2+ gene encodes a protein of a tetratricopeptide repeat (TPR) family which is conserved throughout evolution. We previously showed that nuc2 is required for exit from the mitotic metaphase. In this study, we present evidence which shows that nuc2 has two additional roles in the cell cycle. We showed that the nuc2 mutant is sterile even at the permissive temperature and septation occurs in the absence of chromosome separation at the restrictive temperature. The nuc2 mutant fails to arrest at the G1 phase upon nitrogen starvation at the permissive temperature which is a prerequisite for conjugation. Upon starvation, however, the nuc2 mutant ceased division normally and induced starvation-dependent gene expression. Therefore, the nuc2 mutant is deficient only for failure to block DNA replication upon starvation. At the lower restrictive temperature, the nuc2 mutant showed a ‘cut’ phenotype where septation and cytokinesis takes place without the completion of mitosis. Ectopic overexpression of the nuc2+ gene caused multiple rounds of S and M phases in the complete absence of septum formation. We propose that nuc2 is a novel cell cycle regulator essential for three events; firstly for exit from mitosis, secondly for DNA replication restraint under nutrient starvation and thirdly for inhibition of septation and cytokinesis until the completion of mitosis.

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