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P Nurse

Publications and source records attributed to P Nurse.

At least 19 recordsLinked to original sources
PubMed1997

Fission yeast pheromone blocks S-phase by inhibiting the G1 cyclin B-p34cdc2 kinase.

Yeast pheromones block cell cycle progression in G1 in order to prepare mating partners for conjugation. We have investigated the mechanism underlying pheromone-induced G1 arrest in the fission yeast Schizosaccharomyces pombe. We find that the G1-specific transcription factor p65cdc10-p72res1/sct1 which controls the expression of S-phase genes is fully activated in pheromone, unlike the analogous control in budding yeast. In contrast, the G1 function of p34cdc2 acting after activation of the G1-specific transcription is blocked. Pheromone inhibits the p34cdc2 kinase associated with both the G1-specific B-type cyclin p45cig2 and the B-type cyclin p56cdc13 and overexpression of p45cig2 or p47cdc13delta90 overcomes the pheromone-induced G1 arrest. G1 arrest is compromised in enlarged cells. We suggest that onset of S-phase is controlled by pheromone inhibiting the B-cyclin-associated kinase in G1, and that increasing cell size contributes to the mechanism for pheromone adaptation. Thus, pheromone in fission and budding yeast acts similarly in inhibiting the G1 cyclin-dependent kinase (CDK), but differs in its effects on the G1/S transcriptional control, suggesting that inhibition of CDKs may be a more general mechanism for the control of G1 progression compared with G1/S transcriptional control.

Blotting, Northern↗
PubMed1997

Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation.

The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up-regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.

Blotting, Western↗
PubMed1996

Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein.

We describe a method for identifying genes encoding proteins with stereospecific intracellular localizations in the fission yeast Schizosaccharomyces pombe. Yeast are transformed with a gene library in which S. pombe genomic sequences are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP), and intracellular localizations are subsequently identified by rapid fluorescence screening in vivo. In a model application of these methods to the fission yeast nucleus, we have identified several novel genes whose products are found in specific nuclear regions, including chromatin, the nucleolus, and the mitotic spindle, and sequence similarities between some of these genes and previously identified genes encoding nuclear proteins have validated the approach. These methods will be useful in identifying additional components of the S. pombe nucleus, and further extensions of this approach should also be applicable to a more comprehensive identification of the elements of intracellular architecture in fission yeast.

Amino Acid Sequence↗
PubMed1996

Microtubules mediate mitochondrial distribution in fission yeast.

The Schizosaccharomyces pombe mutant, ban5-4, displays aberrant mitochondrial distribution. Incubation of this conditional-lethal mutant at the nonpermissive temperature led to aggregated mitochondria that were distributed asymmetrically within the cell. Development of this mitochondrial asymmetry but not mitochondrial aggregation required progression through the cell division cycle. Genetic analysis revealed that ban5-4 is an allele of atb2 encoding alpha 2-tubulin. Consistent with this finding, cells with the cold-sensitive nda3 mutation in beta-tubulin displayed aggregated and asymmetrically distributed mitochondria after incubation at lowered temperatures. These results indicate that microtubules mediate mitochondrial distribution in fission yeast and provide the first genetic evidence for the role of microtubules in mitochondrial movement.

Cell Cycle↗
PubMed1996

The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase.

In a screen for new cell-cycle genes in Schizosaccharomyces pombe we have isolated cdc30, which is identical to orp1, a putative homolog of the Saccharomyces cerevisiae ORC1 gene. Analysis of the temperature-sensitive orp1-4 and the orp1(delta) mutants indicates that orp1 is required at the onset of S phase for an early step of DNA replication. Orp1p is found in the nucleus and is present at a constant level throughout the cell cycle. Genetic interactions occur between orp1 and cdc18 and cdc21 (an MCM homolog). Orp1p forms protein complexes with both cdc18p and cdc21p in vivo, suggesting that interactions between these proteins and ORC are important for controlling the initiation of DNA replication at the onset of S phase. The orp1 gene is also required for the control that prevents entry into mitosis in the absence of DNA replication, suggesting a role for ORC in this checkpoint pathway.

Cell Cycle↗
PubMed1996

A novel S phase inhibitor in fission yeast.

We have cloned a novel fission yeast gene, spd1, which causes G1 arrest when overexpressed. Deleting the gene results in cells being accelerated through G1 into S phase in certain circumstances when the G1-->S phase control is compromised. We have found that the encoded 14 kDa protein is cell cycle regulated, declining in level during S phase, and that p14spd1 physically associates with p34cdc2 in vivo when overexpressed, suggesting that p14spd1 may regulate S phase progression via an interaction with p34cdc2. We conclude that p14spd1 is a negative regulator of S phase, and that it may be part of the control ensuring an orderly onset of S phase or part of a G1-->S phase checkpoint control.

Amino Acid Sequence↗
PubMed1996

The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.

Alleles↗
PubMed1996

The ordered assembly of the phiX174-type primosome. III. PriB facilitates complex formation between PriA and DnaT.

The properties of two mutant PriA proteins, PriA C439Y and PriA C445Y have been used to reveal the role of PriB during assembly of the phiX174-type primosome. The replication defects of both mutant PriA proteins could be rescued by high concentrations of DnaT. Analysis of the formation of intermediate complexes in primosome assembly and the effect of PriB on PriA binding to DNA demonstrated that the mutant PriA proteins could not form a PriA-PriB complex on DNA carrying a primosome assembly site. Consequently, the mutant proteins also could not form PriA-PriB-DnaT complexes at concentrations of DnaT sufficient to form such a complex with wild-type PriA. In addition, PriB was found to stabilize wild-type but not mutant PriA proteins on DNA. At high concentrations of DnaT, both mutant and wild-type PriA proteins could form a PriA-DnaT complex and support PriB-independent phiX174 complementary strand DNA replication. Thus, during primosome assembly, PriB facilitates complex formation between PriA and DnaT.

Bacterial Proteins↗
PubMed1996

A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.

CDC2 Protein Kinase↗
PubMed1996

How fission yeast fission in the middle.

In fission yeast, we propose that the division plane may be positioned by the position of the premitotic nucleus, perhaps by a signal emanating from the nucleus. Gene products involved in the assembly of the ring and its temporal and spatial controls are beginning to be characterized. Some of these, such as mid1p, may be involved in signals that position the division site. In animal cells, the division site may be determined by analogous signals emanating from the mitotic asters (Rappaport, 1986). We speculate that the signals defined in fission yeast will help identify signals that determine the division plane in all kinds of eukaryotes.

Cell Division↗
PubMed1996

Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring.

Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a beta-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.

Actins↗
PubMed1996

A quantitative model for the cdc2 control of S phase and mitosis in fission yeast.

In this article we consider the role of the cyclin-dependent protein kinase cdc2 in regulating progression through the fission yeast cell cycle. The onset of mitosis is governed by cdc2 in partnership with the B-type cyclin, cdc13. Recent evidence shows that the cdc2-cdc13 complex can also control the onset of S phase and, in addition, ensures that there is only one S phase per cell cycle. This leads us to propose a novel quantitative model in which different levels of cdc2 activity regulate cell-cycle progression: S phase is initiated when protein kinase activity increases from a very low to a moderate level; maintenance of this moderate level prevents re-initiation of S phase, and a further increase of activity to a high level initiates mitosis. Inactivation of the kinase activity at the end of mitosis resets the cell for a new cell cycle.

Animals↗
PubMed1995

p25rum1 orders S phase and mitosis by acting as an inhibitor of the p34cdc2 mitotic kinase.

p25rum1 from the fission yeast S. pombe is shown to act as a specific in vitro inhibitor of the p34cdc2/p56cdc13 mitotic kinase. It is also shown that early G1 cells contain p25rum1, which associates with and inhibits the mitotic kinase, and maintains p56cdc13 mitotic B cyclin at a low level, ensuring that these cells do not undergo a premature lethal entry into mitosis. A high level of p25rum1 in G2 cells inhibits the p34cdc2/p56cdc13 kinase that removes the block preventing a further S phase and leads to repeated rounds of DNA replication. Thus, the cyclin-dependent kinase inhibitor p25rum1, acting on the p34cdc2 mitotic kinase, plays an important role in ensuring the correct sequence of S phase and mitosis during the cell cycle.

CDC2 Protein Kinase↗
PubMed1995

p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast.

A key problem in the cell cycle is understanding what brings about the initiation of DNA replication and how this is linked with global cell cycle controls. The fission yeast gene cdc18 is required for DNA replication and is transcriptionally activated by the cdc10/res1/res2 control acting at START in late G1. We show here that overexpressing cdc18 is able to bring about repeated rounds of DNA synthesis in the absence of mitosis and of continuing protein synthesis. The level of the cdc18-encoded protein p65cdc18 is periodic in the cell cycle, peaking at the G1 to S phase transition, and p65cdc18 is located in the nucleus when cdc18 is overexpressed. We propose that p65cdc18 acts at the initiation of DNA replication and plays a major role in controlling the onset of S phase.

Cell Cycle Proteins↗
PubMed1995

A pre-start checkpoint preventing mitosis in fission yeast acts independently of p34cdc2 tyrosine phosphorylation.

We have monitored the tyrosine (Y15) phosphorylated and dephosphorylated forms of p34cdc2 from Schizosaccharomyces pombe as cells proceed through the cell cycle. Y15 is dephosphorylated in G1 before start and becomes phosphorylated only after cells pass start and enter late G1. This transition is associated with a switch from one checkpoint which restrains mitosis in pre-start G1, by a mechanism independent from Y15 phosphorylation, to a second checkpoint acting post-start during late G1 and S phase operating through Y15 phosphorylation. The pre-start checkpoint may act by preventing formation of the p34cdc2/p56cdc13 complex. The complex between Y15-phosphorylated p34cdc2 and p56cdc13 accumulates during S phase and G2, but the level generated is not solely dependent on the amount of p34cdc2 and p56cdc13 present in the cell. The extent of p56cdc13 breakdown at the end of mitosis may be determined by the amount complexed with p34cdc2. We have also shown that an insoluble form of p34cdc2 is associated with the progression of the cell through late G1 into S phase.

Antibodies, Fungal↗
PubMed1995

Dominant mutants identify new roles for p34cdc2 in mitosis.

A large number of dominant mutants have been generated in the fission yeast cdc2 gene, causing lethality when expressed in wild-type cells. The mutants interfere with distinct aspects of p34cdc2 function, producing one of four different phenotypes: mitotic arrest, multiple rounds of S phase in the absence of mitosis, premature mitosis or G2 arrest. The mitotic mutants DL41, DL45 and DL50 are characterized in this paper. Over-expression of DL41 or DL45 causes mitotic arrest, specifically interfering with sister chromatid separation, without preventing spindle elongation. This suggests a role for p34cdc2 in triggering sister chromatid separation at anaphase. DL41 and DL45 also cause abnormal septum formation, suggesting that p34cdc2 may also be involved in regulating this process in fission yeast. These mitotic aspects of p34cdc2 function may involve interaction with p13suc1, since increased expression of suc1 partially suppresses DL41 and DL45. Over-expression of DL50 causes premature mitotic entry in cells that have not completed S phase, resulting in lethality. DL41, DL45 and DL50 correspond to mutation of p34cdc2 residues predicted to be on the surface of the protein, identifying potential sites of interaction with mitotic regulators of p3cdc2, and these residues are conserved amongst cdc2 proteins found in other eukaryotes.

CDC2 Protein Kinase↗
PubMed1995

The cell cycle and suc1: from structure to function?

Structures have recently been determined for the yeast Schizosaccharomyces pombe cell cycle regulatory protein, CKS/suc1, and its human equivalent. The structures provide some long-awaited clues about the role of CKS/suc1 in cell cycle control.

Binding Sites↗