SGV1 encodes a CDC28/cdc2-related kinase required for a G alpha subunit-mediated adaptive response to pheromone in S. cerevisiae.

The GPA1 gene of S. cerevisiae encodes a G alpha subunit that plays a positive role in the transduction of signals stimulating recovery from pheromone-induced cell cycle arrest. The GPA1Val50 mutation, in which Gly-50 is replaced by valine, causes hyperadaptation to pheromone. However, GPA1Val50 cells do not recover from division arrest in the absence of both CLN1 and CLN3, which encode G1 cyclins, indicating that the recovery-promoting activity of GPA1Val50 requires the function of G1 cyclins. An sgv1 mutation suppresses the hyperadaptive response caused by GPA1Val50 and also confers cold- and temperature-sensitive growth. The SGV1 gene encodes an apparent protein kinase homologous to CDC28/cdc2 kinase: SGV1 is 42% identical to CDC28. The activated mutation, CLN3-2, partially suppresses the growth defect of sgv1, suggesting that the SGV1 and CLN3 proteins may act in the same growth control pathway.     

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.     

A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle

The CLN1, CLN2, and CLN3 genes of S. cerevisiae form a redundant family essential for the G1-to-S phase transition. CLN1 and CLN2 mRNAs were previously shown to be negatively regulated by mating pheromone and by cell cycle progression out of G1, whereas CLN3 mRNA is not. The CLN3-2 (DAF1-1) allele prevents both cell cycle arrest and the turnoff of CLN1 and CLN2 mRNAs in response to mating pheromone, but only in the presence of an active CDC28 gene. An internally deleted nonfunctional cln2 gene was used as a reporter gene to demonstrate that in the absence of mating pheromone, efficient expression of cln2 mRNA requires both an active CDC28 gene and at least one functional CLN gene. mRNA from a nonfunctional cln1 gene was regulated similarly. Thus, CLN function and CDC28 activity jointly stimulate CLN1 and CLN2 mRNA levels, potentially forming a positive feedback loop for CLN1 and CLN2 expression.     

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.     

SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities.

Traversal from G1 to S-phase in cycling cells of budding yeast is dependent on the destruction of the S-phase cyclin/CDK inhibitor SIC1. Genetic data suggest that SIC1 proteolysis is mediated by the ubiquitin pathway and requires the action of CDC34, CDC4, CDC53, SKP1, and CLN/CDC28. As a first step in defining the functions of the corresponding gene products, we have reconstituted SIC1 multiubiquitination in DEAE-fractionated yeast extract. Multiubiquitination depends on cyclin/CDC28 protein kinase and the CDC34 ubiquitin-conjugating enzyme. Ubiquitin chain formation is abrogated in cdc4ts mutant extracts and assembly restored by the addition of exogenous CDC4, suggesting a direct role for this protein in SIC1 multiubiquitination. Deletion analysis of SIC1 indicates that the N-terminal 160 residues are both necessary and sufficient to serve as substrate for CDC34-dependent ubiquitination. The complementary C-terminal segment of SIC1 binds to the S-phase cyclin CLB5, indicating a modular structure for SIC1.     

High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6

ObjectivesCDC45 is the core component of CMG (CDC45‐MCMs‐GINS) complex that plays important role in the initial step of DNA replication in eukaryotic cells. The expression level of cdc45 is under the critical control for the accurate cell cycle progression. Loss‐of‐function of cdc45 has been demonstrated to inhibit cell proliferation and leads to cell death due to the inhibition of DNA replication and G1‐phase arrest. An increasing of CDC45 inhibits cell proliferation as well. Nevertheless, a systematic analysis of the effect of high dose of CDC45 on cell physiology and behaviors is unclear. In the present study, we aimed to investigate the effects and mechanisms of high dose of CDC45 on cell behaviors.Materials and MethodsWe overexpressed cdc45 in cultured cell lines, Ciona and Drosophila embryos, respectively. The cell cycle progression was examined by the BrdU incorporation experiment, flow cytometry and PH3 (phospho‐Histone 3) staining. RNA‐sequencing analysis and qRT‐PCR were carried out to screen the affected genes in HeLa cells overexpressing cdc45. siRNA‐mediated knockdown was performed to investigate gene functions in HeLa cells overexpressing cdc45.ResultsWe found that high level of cdc45 from different species (human, mammal, ascidian, and Drosophila) inhibited cell cycle in vitro and in vivo. High dose of CDC45 blocks cells entering into S phase. However, we failed to detect DNA damage and cell apoptosis. We identified hspa6 was the most upregulated gene in HeLa cells overexpressing cdc45 via RNA‐seq analysis and qRT‐PCR validation. Overexpression of Hs‐hspa6 inhibited proliferation rate and DNA replication in HeLa cells, mimicking the phenotype of cdc45 overexpression. RNAi against hspa6 partially rescued the cell proliferation defect caused by high dose of CDC45.ConclusionsOur study suggests that high abundance of CDC45 stops cell cycle. Instead of inducing apoptosis, excessive CDC45 prevents cell entering S phase probably due to promoting hspa6 expression.

CDC45 is essential for DNA replication. Surprisingly high dose of CDC45 inhibits cell proliferation and blocks cell entering S phase without inducing apoptosis nor aneuploidy as expected. The overexpressed CDC45 induces the elevation of HSPA6, which in turn inhibits cell proliferation.     

Yeast cell death during DNA damage arrest is independent of caspase or reactive oxygen species

CDC13 encodes a telomere-binding protein that prevents degradation of telomeres. cdc13-1 yeast grown at the nonpermissive temperature undergo G2/M arrest, progressive chromosome instability, and subsequent cell death. Recently, it has been suggested that cell death in the cdc13-1 mutant is an active process characterized by phenotypic hallmarks of apoptosis and caspase activation. In this work, we show that cell death triggered by cdc13-1 is independent of the yeast metacaspase Yca1p and reactive oxygen species but related to cell cycle arrest per se. Inactivating YCA1 or depleting reactive oxygen species does not increase viability of cdc13-1 cells. In turn, caspase activation does not precede cell death in the cdc13-1 mutant. Yca1p activity assayed by cell binding of mammalian caspase inhibitors is confounded by artifactual labeling of dead yeast cells, which nonspecifically bind fluorochromes. We speculate that during a prolonged cell cycle arrest, cdc13-1 cells reach a critical size and die by cell lysis.     

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.     

Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2val19 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also show that intracellular acidification stimulates the pathway at or upstream of the CDC25 protein. When shifted to the restrictive temperature, cells with the temperature sensitive cdc25-5 mutation lost their cAMP content within a few minutes. After prolonged incubation at the restrictive temperature, cells with this mutation, and also those with the temperature sensitive cdc25-1 mutation, arrested at the 'start' point (in G1) of the cell cycle, and subsequently accumulated in the resting state G0. In contrast with cdc25-5 cells, however, the cAMP level did not decrease and normal glucose- and acidification-induced cAMP responses were observed when cdc25-1 cells were shifted to the restrictive temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Cdc20, a β-transducin homologue,links RAD9-mediated G2/M checkpoint control to mitosis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the DNA damage-induced G2 arrest requires the checkpoint control genes RAD9, RAD17, RAD24, MEC1, MEC2 and MEC3. These genes also prevent entry into mitosis of a temperature-sensitive mutant, cdc13, that accumulates chromosome damage at 37^°?C. Here we show that a cdc13 mutant overexpressing Cdc20, a β-transducin homologue, no longer arrests in G2 at the restrictive temperature but instead undergoes nuclear division, exits mitosis and enters a subsequent division cycle, which suggests that the DNA damage-induced G2/M checkpoint control is not functional in these cells. This is consistent with our observation that overexpression of CDC20 in wild-type cells results in increased sensitivity to UV irradiation. Overproduction of Cdc20 does not influence the arrest phenotype of the cdc mutants whose cell cycle block is independent of RAD9-mediated checkpoint control. Therefore, we suggest that the DNA damage-induced checkpoint controls prevent mitosis by inhibiting the nuclear division pathway requiring CDC20 function.     

Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

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/G(2) 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 G(2) 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/G(2) phase.     

Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats.

We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitive [corrected] defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras. In contrast to the yeast RAS genes, Xenopus N-ras rescues the cdc15 mutation. Moreover, overexpression of Xenopus N-ras in S. cerevisiae does not activate the RAS-cyclic AMP (cAMP) pathway; rather, it results in decreased levels of intracellular cAMP in both mutant cdc15 and wild-type cells. Furthermore, we show that lowering cAMP levels is sufficient to allow cells with a nonfunctional Cdc15 protein to complete the mitotic cycle. These results suggest that a key step of the cell cycle is dependent upon a phosphorylation event catalyzed by cAMP-dependent protein kinase. The second clone, beta TrCP (beta-transducin repeat-containing protein), encodes a protein of 518 amino acids that shows significant homology to the beta subunits of G proteins in its C-terminal half. In this region, beta Trcp is composed of seven beta-transducin repeats. beta TrCP is not a functional homolog of S. cerevisiae CDC20, a cell cycle gene that also contains beta-transducin repeats and suppresses the cdc15 mutation.