Reduced dosage of a single fission yeast MCM protein causes genetic instability and S phase delay

1999 ◽  
Vol 112 (4) ◽  
pp. 559-567 ◽  
Author(s):  
D.T. Liang ◽  
J.A. Hodson ◽  
S.L. Forsburg
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Replication Initiation ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Protein Levels ◽  
Mcm Proteins

MCM proteins are a conserved family of eukaryotic replication factors implicated in the initiation of DNA replication and in the discrimination between replicated and unreplicated chromatin. However, most mcm mutants in yeast arrest the cell cycle after bulk DNA synthesis has occurred. We investigated the basis for this late S phase arrest by analyzing the effects of a temperature-sensitive mutation in fission yeast cdc19(+)(mcm2(+)). cdc19-P1 cells show a dramatic loss of viability at the restrictive temperature, which is not typical of all S phase mutants. The cdc19-P1 cell cycle arrest requires an intact damage-response checkpoint and is accompanied by increased rates of chromosome loss and mitotic recombination. Chromosomes from cdc19-P1 cells migrate aberrantly in pulsed-field gels, typical of strains arrested with unresolved replication intermediates. The cdc19-P1 mutation reduces the level of the Cdc19 protein at all temperatures. We compared the effects of disruptions of cdc19(+)(mcm2(+)), cdc21(+)(mcm4(+)), nda4(+)(mcm5(+)) and mis5(+)(mcm6(+)); in all cases, the null mutants underwent delayed S phase but were unable to proceed through the cell cycle. Examination of protein levels suggests that this delayed S phase reflects limiting, but not absent, MCM proteins. Thus, reduced dosage of MCM proteins allows replication initiation, but is insufficient for completion of S phase and cell cycle progression.

scholarly journals Functions of Fission Yeast Orp2 in DNA Replication and Checkpoint Control

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 599-607
Author(s):  
Joan Kiely ◽  
S B Haase ◽  
Paul Russell ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Control ◽  
Cycle Arrest

Abstract orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.

Cloning of a human S-phase cell cycle gene: use of transient expression for screening

1987 ◽  
Vol 7 (2) ◽  
pp. 775-779
Author(s):  
A Fainsod ◽  
G Diamond ◽  
M Marcus ◽  
F H Ruddle
Keyword(s):  
Cell Cycle ◽  
Genomic Library ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
S Phase ◽  
Phase Cell ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Restrictive Temperature

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

scholarly journals MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae.

1993 ◽  
Vol 123 (2) ◽  
pp. 387-403 ◽  
Author(s):  
M T Brown ◽  
L Goetsch ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
In Vitro Mutagenesis ◽  
Spindle Elongation

The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.

Caffeine can override the S-M checkpoint in fission yeast

1999 ◽  
Vol 112 (6) ◽  
pp. 927-937 ◽  
Author(s):  
S.W. Wang ◽  
C. Norbury ◽  
A.L. Harris ◽  
T. Toda
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
S Phase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Control ◽  
Animal Cells ◽  
Replication Checkpoint ◽  
Phase Arrest ◽  
S Phase Arrest

The replication checkpoint (or ‘S-M checkpoint’) control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control.

scholarly journals Fission Yeast rad12+Regulates Cell Cycle Checkpoint Control and Is Homologous to the Bloom’s Syndrome Disease Gene

1998 ◽  
Vol 18 (5) ◽  
pp. 2721-2728 ◽  
Author(s):  
Scott Davey ◽  
Christine S. Han ◽  
Sarah A. Ramer ◽  
Jennifer C. Klassen ◽  
Adam Jacobson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Uv Light ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Checkpoint Control ◽  
Bloom's Syndrome ◽  
Synthesis Inhibitor ◽  
Bloom’S Syndrome

ABSTRACT The human BLM gene is a member of the Escherichia coli recQ helicase family, which includes the Saccharomyces cerevisiae SGS1 and human WRN genes. Defects inBLM are responsible for the human disease Bloom’s syndrome, which is characterized in part by genomic instability and a high incidence of cancer. Here we describe the cloning ofrad12 +, which is the fission yeast homolog ofBLM and is identical to the recently reportedrhq1 + gene. We showed that rad12null cells are sensitive to DNA damage induced by UV light and γ radiation, as well as to the DNA synthesis inhibitor hydroxyurea. Overexpression of the wild-type rad12 + gene also leads to sensitivity to these agents and to defects associated with the loss of the S-phase and G2-phase checkpoint control. We showed genetically and biochemically thatrad12 + acts upstream fromrad9 +, one of the fission yeast G2checkpoint control genes, in regulating exit from the S-phase checkpoint. The physical chromosome segregation defects seen inrad12 null cells combined with the checkpoint regulation defect seen in the rad12 + overproducer implicate rad12 + as a key coupler of chromosomal integrity with cell cycle progression.

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

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.

scholarly journals Characterization ofSaccharomyces cerevisiae dna2Mutants Suggests a Role for the Helicase Late in S Phase

1997 ◽  
Vol 8 (12) ◽  
pp. 2519-2537 ◽  
Author(s):  
David F. Fiorentino ◽  
Gerald R. Crabtree
Keyword(s):  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Temperature Shift ◽  
Synthesis Temperature ◽  
Kinase Domain ◽  
S Phase ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Dependent Manner

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like “lipid kinase” domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical toDNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality withtor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- andMEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.

scholarly journals Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

1987 ◽  
Vol 7 (2) ◽  
pp. 775-779 ◽  
Author(s):  
A Fainsod ◽  
G Diamond ◽  
M Marcus ◽  
F H Ruddle
Keyword(s):  
Cell Cycle ◽  
Genomic Library ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
S Phase ◽  
Phase Cell ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Restrictive Temperature

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

scholarly journals Characterization of Schizosaccharomyces pombe mcm7+ and cdc23+ (MCM10) and Interactions With Replication Checkpoints

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 471-486 ◽  
Author(s):  
Debbie T Liang ◽  
Susan L Forsburg
Keyword(s):  
Fission Yeast ◽  
S Phase ◽  
The Other ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
2C Dna Content ◽  
Mcm Proteins ◽  
Sensitive Allele ◽  
Synthetically Lethal ◽  
Yeast Homolog

Abstract MCM proteins are required for the proper regulation of DNA replication. We cloned fission yeast mcm7+ and showed it is essential for viability; spores lacking mcm7+ begin S phase later than wild-type cells and arrest with an apparent 2C DNA content. We isolated a novel temperature-sensitive allele, mcm7-98, and also characterized two temperature-sensitive alleles of the fission yeast homolog of MCM10, cdc23+. mcm7-98 and both cdc23ts alleles arrest with damaged chromosomes and an S phase delay. We find that mcm7-98 is synthetically lethal with the other mcmts mutants but does not interact genetically with either cdc23ts allele. However, cdc23-M36 interacts with mcm4ts. Unlike other mcm mutants or cdc23, mcm7-98 is synthetically lethal with checkpoint mutants Δcds1, Δchk1, or Δrad3, suggesting chromosomal defects even at permissive temperature. Mcm7p is a nuclear protein throughout the cell cycle, and its localization is dependent on the other MCM proteins. Our data suggest that the Mcm3p-Mcm5p dimer interacts with the Mcm4p-Mcm6p-Mcm7p core complex through Mcm7p.

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