The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis

1995 ◽  
Vol 108 (2) ◽  
pp. 475-486 ◽  
Author(s):  
F. al-Khodairy ◽  
T. Enoch ◽  
I.M. Hagan ◽  
A.M. Carr
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Control ◽  
S Phase ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Efficient Recovery ◽  
Mediate Cell

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘checkpoints’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

The Effect of 2-Phenyl Ethanol on the DNA Synthesis Cycle of Schizosaccharomyces Pombe

1970 ◽  
Vol 7 (2) ◽  
pp. 523-530
Author(s):  
C. J. BOSTOCK
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Normal Growth ◽  
S Phase ◽  
G1 Phase ◽  
Synchronous Cultures ◽  
Number Of Cells

The effect of different concentrations of 2-phenyl ethanol (PE) on growth and DNA synthesis of Schizosaccharomyces pombe is described. o.3% PE inhibits the entry of cells into S phase, but allows a doubling in the number of cells in the culture. The effect of o.2% PE on random and synchronous cultures of S. pombe shows that, in the continued presence of the inhibitor, the S phase is moved to a different point in the cell cycle. Cells continue to grow in the presence of o.2% PE with a G1 phase occupying a significant portion of the cell cycle. This differs from normal growth when the G1 phase is absent.

scholarly journals Mcs4, a Two-Component System Response Regulator Homologue, Regulates the Schizosaccharomyces pombe Cell Cycle Control

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1043-1051 ◽  
Author(s):  
Guillaume Cottarel
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Response Regulator ◽  
Cell Cycle Control ◽  
Mitotic Catastrophe ◽  
System Response ◽  
Temperature Sensitive ◽  
Two Component System ◽  
Component System ◽  
Two Component

The Schizosaccharomyces pombe cdc2-3w weel-50 double mutant displays a temperature-sensitive lethal phenotype termed mitotic catastrophe. Six mitotic catastrophe suppressor (mcs1-6) genes were identified in a genetic screen designed to identify regulators of cdc2. Mutations in mcs1-6 suppress the cdc2-3w weel-50 temperature-sensitive growth defect. Here, the cloning of mcs4 is described. The mcs4 gene product displays significant sequence homology to members of the two-component system response regulator protein family. Strains carrying the mcs4 and cdc25 mutations display a synthetic osmotic lethal phenotype along with an inability to grow on minimal synthetic medium. These phenotypes are suppressed by a mutation in wee1. In addition, the wis1 gene, encoding a stress-activated mitogen-activated protein kinase kinase, was identified as a dosage suppressor in this screen. These findings link the two-component signal transduction system to stress response and cell cycle control in S. pombe.

A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints

2000 ◽  
Vol 113 (6) ◽  
pp. 1075-1088
Author(s):  
D. Griffiths ◽  
M. Uchiyama ◽  
P. Nurse ◽  
T.S. Wang
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Radiation Sensitivity ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dna Polymerase Delta ◽  
Synthesis Inhibitor ◽  
Cycle Arrest ◽  
Checkpoint Pathway ◽  
Higher Doses

To further dissect the genetic differences between the checkpoint pathway following S-phase cdc arrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase delta mutant, cdc20-M10. One such checkpoint mutant, rad17-d14, was found to display the cut phenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1 mutant. Unlike chk1, rad17-d14 was not sensitive to UV irradiation. Interestingly, the ionising radiation sensitivity of rad17-d14 was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G(2) phase. Biochemical analysis attributes the checkpoint defects of rad17-d14 to the failure to phosphorylate the checkpoint effector Chk1p. To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed. Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase. This interaction occurred irrespective of the arrest with cdc20-M10 and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.

DNA polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast

1995 ◽  
Vol 108 (9) ◽  
pp. 3109-3118 ◽  
Author(s):  
G. D'Urso ◽  
B. Grallert ◽  
P. Nurse
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
S Phase ◽  
Replication Initiation ◽  
Direct Immunofluorescence ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Initiation Complex ◽  
Dna Polymerase Alpha ◽  
Checkpoint Signal

Genetic analysis in the yeast Schizosaccharomyces pombe has shown that three genes cdc18, cut5, and cdt1, are essential for DNA synthesis and also for the checkpoint control that couples completion of DNA replication to the onset of mitosis. To test whether assembly of the replication initiation complex is an important element in the checkpoint control pathway we have investigated if DNA polymerase alpha (pol1), a component of the initiation complex, is essential for the S-phase checkpoint control. We show that germinating S. pombe spores disrupted for the pol1 gene enter mitosis despite defects in DNA synthesis. This is shown by monitoring septation index, DNA content, and by direct immunofluorescence of mitotic spindles using antibodies to alpha-tubulin. In addition we have isolated six temperature sensitive mutants in the pol1 gene that cause cell cycle arrest when grown at the nonpermissive temperature. Our experiments support a model in which DNA polymerase alpha, in addition to being part of the initiation complex, is required for a checkpoint signal that is activated as cells traverse START, and is essential to prevent mitosis until S phase has been completed. In contrast, proteins responsible for the elongation of DNA may not be necessary for this checkpoint signal.

scholarly journals Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
Author(s):  
Xuan Wang ◽  
Grzegorz Ira ◽  
José Antonio Tercero ◽  
Allyson M. Holmes ◽  
John F. X. Diffley ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Dna Ligases ◽  
Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

scholarly journals The Prpl  + Gene Required for Pre-mRNA Splicing in Schizosaccharomyces pombe Encodes a Protein That Contains TPR Motifs and Is Similar to Prp6p of Budding Yeast

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 101-115 ◽  
Author(s):  
Seiichi Urushiyama ◽  
Tokio Tani ◽  
Yasumi Ohshima
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Cell Cycle Control ◽  
Mrna Splicing ◽  
Restrictive Temperature

Abstract The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)+ RNA transport. The prp1  + gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prplp shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1  + and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1  + gene was found to be identical with the zer1  + gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)+ RNA nuclear export, in addition to pre-mRNA splicing.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe

1986 ◽  
Vol 6 (10) ◽  
pp. 3523-3530
Author(s):  
R Booher ◽  
D Beach
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Cell Cycle Control ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Atp Binding Site ◽  
Leu2 Gene ◽  
Cdc2 Gene ◽  
A Site

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.

Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5710-5717
Author(s):  
E A Malone ◽  
C D Clark ◽  
A Chiang ◽  
F Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Copy Number ◽  
Cell Cycle Control ◽  
Control Point ◽  
Temperature Sensitive ◽  
Upstream Activating Sequence ◽  
In Trans ◽  
Cis And Trans ◽  
Insertion Mutations

SPT16 was previously identified as a high-copy-number suppressor of delta insertion mutations in the 5' regions of the HIS4 and LYS2 genes of Saccharomyces cerevisiae. We have constructed null mutations in the SPT16 gene and have demonstrated that it is essential for growth. Temperature-sensitive-lethality spt16 alleles have been isolated and shown to be pleiotropic; at a temperature permissive for growth, spt16 mutations suppress delta insertion mutations, a deletion of the SUC2 upstream activating sequence, and mutations in trans-acting genes required for both SUC2 and Ty expression. In addition, SPT16 is identical to CDC68, a gene previously shown to be required for passage through the cell cycle control point START. However, at least some transcriptional effects caused by spt16 mutations are independent of arrest at START. These results and those in the accompanying paper (A. Rowley, R. A. Singer, and G. C. Johnston, Mol. Cell. Biol. 11:5718-5726, 1991) indicate that SPT16/CDC68 is required for normal transcription of many loci in S. cerevisiae.

scholarly journals The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

1997 ◽  
Vol 17 (5) ◽  
pp. 2381-2390 ◽  
Author(s):  
A E Parker ◽  
R K Clyne ◽  
A M Carr ◽  
T J Kelly
Keyword(s):  
Dna Repair ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Excision Repair ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

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