Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants

Abstract The temperature-sensitive dna mutants of the budding yeast Saccharomyces cerevisiae (Dumas et al. (1982) Mol. Gen. Genet. 187, 42–46) are more inhibited in DNA synthesis than in protein synthesis. These properties are also characteristics of many yeast mutations that inhibit progress through the cell cycle. Therefore we surveyed the collection of dna mutants for cell-cycle mutations. By genetic complementation we found that dna 1 = cdc 22, dna 6 = cdc 34, dna 19 = cdc 36, and dna 39 = dbf 3. Furthermore, by direct gene cloning we found that the dna26 mutation is allelic to prt1 mutations, which are known to exert primary inhibition on protein synthesis. This protein-synthesis mutation exerts a dna phenotype due to cell-cycle inhibition: prt1 mutations can block the regulatory step of the cell cycle while allowing significant amounts of protein synthesis to continue. Our non-exhausive screening suggests that the dna mutants may house other mutations that affect the yeast cell cycle.     

Genetic studies with the fission yeast Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr

Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G(2)/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast Schizosaccharomyces pombe, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (cdc phenotype) typical of G(2)/M cell cycle arrest in wild-type and various mutant strains of S. pombe was examined. Our results indicated that Vpr caused the cdc phenotype in wild-type S. pombe as well as in strains carrying mutations, such as the cdc2-3w, Deltacdc25, rad1-1, Deltachk1, Deltamik1, and Deltappa1 strains. However, other mutants, such as the cdc2-1w, Deltawee1, Deltappa2, and Deltarad24 strains, failed to show a distinct cdc phenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the cdc phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.     

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.     

Mapping CDC Mutations in the Yeast S. Cerevisiae by RAD52-Mediated Chromosome Loss

Using the chromosome loss-mapping method of Schild and Mortimer, I have mapped several new temperature-sensitive mutations that define five CDC genes. Modified procedures were used to facilitate mapping temperature-sensitive mutations in general, and these modifications are discussed. The mutations were assigned to specific chromosomes by chromosome loss procedures, and linkage relationships were determined subsequently by standard tetrad analysis. Four of the mutations define new loci. The fifth mutation, cdc63-1, is shown to be allelic to previously known mutations in the PRT1 gene.     

Specific early-G1 blocks accompanied with stringent response in Saccharomyces cerevisiae lead to growth arrest in resting state similar to the G0 of higher eucaryotes

Growth arrests of Saccharomyces cerevisiae cells in early G1 phase brought by various means were classified into two types according to the mode of growth recovery after release of the restraints against growth. The first type, including arrests caused by cdc25, cdc33, cdc35, and ils1 mutations at the nonpermissive temperature and also by sulfur starvation, showed a subsequent delay in the onset of budding when shifted back to permissive conditions. The length of the delay was positively correlated with the time that cells had been arrested. The second type, including those caused by cdc28 and cdc24 mutations and by alpha factor, did not affect the mode of growth recovery after the shift to permissive conditions irrespective of the time that cell proliferation had been restricted. Growth arrests of the first type seem to allow yeast cells to enter a resting state equivalent to the G0 state of higher eucaryotes because features of the G0 shown with lymphocytes and other cultured cells including unusually long delay before the growth recovery (L.H. Augenlicht and R. Baserga, 1974, Exp. Cell Res., 89:255-262; and Kumagai, J., H. Akiyama, S. Iwashita, H. lida, and I. Yahara, 1981, J. Immunol., 126:1249-1254) appeared to be associated with this type. We have noted that arrests of the first type were always accompanied with a stringent response of macromolecular synthesis and its partial release by cycloheximide. Mapping of arrest points along the path of the cell cycle by the reciprocal shift experiment suggested that arrest points in G1 that led to the G0-like arrest precede or are near the step sensitive to alpha-factor.     

Bud formation by the yeast Saccharomyces cerevisiae is directly dependent on "start"

Cells of the yeast Saccharomyces cerevisiae, which bear a cdc4 gene mutation, arrest early in the cell cycle but continue to produce buds in a periodic fashion. We show here that this periodic bud formation by cells already arrested at the CDC4 step is inhibited if the cell cycle regulatory step "start" is also specifically blocked by mutation or by the presence of the yeast mating pheromone alpha-factor. Thus, the characteristic periodic bud formation by cdc4 mutant cells requires the continued ability to perform start. This finding raises questions concerning the nature of start; these issues are discussed.     

CDC44: a putative nucleotide-binding protein required for cell cycle progression that has homology to subunits of replication factor C.

To investigate the means by which a cell regulates the progression of the mitotic cell cycle, we characterized cdc44, a mutation that causes Saccharomyces cerevisiae cells to arrest before mitosis. CDC44 encodes a 96-kDa basic protein with significant homology to a human protein that binds DNA (PO-GA) and to three subunits of human replication factor C (also called activator 1). The hypothesis that Cdc44p is involved in DNA metabolism is supported by the observations that (i) levels of mitotic recombination suggest elevated rates of DNA damage in cdc44 mutants and (ii) the cell cycle arrest observed in cdc44 mutants is alleviated by the DNA damage checkpoint mutations rad9, mec1, and mec2. The predicted amino acid sequence of Cdc44p contains GTPase consensus sites, and mutations in these regions cause a conditional cell cycle arrest. Taken together, these observations suggest that the essential CDC44 gene may encode the large subunit of yeast replication factor C.     

Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.     

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.     

Cdc42p GTPase is involved in controlling polarized cell growth in Schizosaccharomyces pombe.

Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis. We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-1ts mutation. S. pombe Cdc42p is 85% identical in predicted amino acid sequence to S. cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog. The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools. We have disrupted the cdc42+ gene and shown that it is essential for growth. The cdc42 null phenotype is an arrest as small, round, dense cells. In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S. cerevisiae CDC42. In contrast to the S. cerevisiae cdc42 mutations, the S. pombe cdc42 mutant alleles were not lethal when overexpressed. However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S. pombe Cdc42p is involved in controlling polarized cell growth.     

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold- sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase- encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.     

Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition. © 1994 Wiley-Liss, Inc.     

Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect

The PKC1 gene of Saccharomyces cerevisiae encodes a homologue of the Ca(2+)-dependent isozymes of mammalian protein kinase C (Levin, D.cE., F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner. 1990. Cell. 62:213-224). Cells depleted of the PKC1 gene product display a uniform phenotype, a behavior indicating a defect in the cell division cycle (cdc). These cells arrest division after DNA replication, but before mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrest growth with small buds. We created conditional alleles of PKC1 to explore the nature of this unusual cdc defect. In contrast to PKC1-depleted cells, all of the conditional pkc1 mutants isolated were suppressed by the addition of CaCl2 to the medium, suggesting that the mutant enzymes could be activated by Ca2+. Arrest of growth and cell division in the conditional mutants was accompanied by cessation of protein synthesis, rapid loss of viability, and release of cellular material into the medium, suggesting cell lysis. This conclusion was supported by the observation that a pkc1 deletion mutant was capable of proliferation in osmotically stabilized medium, but underwent rapid cell lysis when shifted to hypo-osmotic medium. We have incorporated these observations into a model to explain the cdc-specific arrest of pkc1 mutants.     

Rise in intracellular pH is concurrent with 'start' progression of Saccharomyces cerevisiae

Intracellular pH (pHi) was determined during arrest and recovery of temperature sensitive-cell division cycle mutants of Saccharomyces cerevisiae. In all mutants, pHi decreased during arrest; but when the mutants were released from arrest a rapid increase in pHi ensued in only cdc28- and cdc37-arrested cells. Both of these mutations cause arrest at 'start', the sole regulatory point in the S. cerevisiae cell cycle. In cells with cdc4 or cdc7 mutations, which arrest past start, pHi remained constant and exhibited a decrease, respectively, upon recovery of growth. The activity of plasma membrane ATPase decreased during the first 30 min of recovery of cdc28-arrested cells, concomitant with the rise in pHi. During the same period, there was no significant change in activity in cdc4-bearing cells, whereas an increase was observed for cdc7-bearing cells. Increase in pHi may be used as a specific signal by S. cerevisiae for start traversal and commitment to a new cycle.     

The Saccharomyces recombination protein Tid1p is required for adaptation from G2/M arrest induced by a double-strand break

Saccharomyces cells with a single unrepaired double-strand break (DSB) will adapt to checkpoint-mediated G2/M arrest and resume cell cycle progression. The decision to adapt is finely regulated by the extent of single-stranded DNA generated from a DSB. We show that cells lacking the recombination protein Tid1p are unable to adapt, but that this defect is distinct from any role in recombination. As with the adaptation-defective mutations yku70Delta and cdc5-ad, permanent arrest in tid1Delta is bypassed by the deletion of the checkpoint gene RAD9. Permanent arrest of tid1Delta cells is suppressed by the rfa1-t11 mutation in the ssDNA binding complex RPA, similar to yku70Delta, whereas the defect in cdc5-ad is not suppressed. Unlike yku70Delta, tid1Delta does not affect 5'-to-3' degradation of DSB ends. The tid1Delta defect cannot be complemented by overexpressing the homolog Rad54p, nor is it affected in rad51Delta tid1Delta, rad54Delta tid1Delta, or rad52Delta tid1Delta double mutants that prevent essentially all homologous recombination. We suggest that Tid1p participates in monitoring the extent of single-stranded DNA produced by resection of DNA ends in a fashion that is distinct from its role in recombination.