A Rad9-Dependent Checkpoint Blocks Meiosis of Cdc13 Yeast Cells

Mutations in CDC13 have previously been found to cause cell cycle arrest of Saccharomyces cerevisiae at a stage in G2 immediately preceding the mitotic division. We show here that cdc13 blocks the meiotic pathway at a stage that follows DNA replication, but in this case the spindle has not yet formed nor have the chromosomes undergone synapsis or recombination. This arrest is alleviated by rad9, thus implicating the same checkpoint function that delays mitotic progression when chromosomal lesions are present. An assessment of the spores produced upon alleviation of the meiotic arrest by rad9 reveals that the absence of recombination in strains bearing cdc13 alone is attributable to the RAD9-mediated arrest rather than to other effects of cdc13 lesions. We have tested the possibility that this checkpoint function is important in regulating meiotic progression to permit resolution of recombinational intermediates during ongoing meiosis and have found no evidence that rad9 alters the execution of functions that might depend upon such regulation. We consider the possible role of other checkpoints in yeast meiosis.     

A dependent pathway of gene functions leading to chromosome segregation in Saccharomyces cerevisiae

Methyl-benzimidazole-2-ylcarbamate (MBC) inhibits the mitotic cell cycle of Saccharomyces cerevisiae at a stage subsequent to DNA synthesis and before the completion of nuclear division (Quinlan, R. A., C. I. Pogson, and K, Gull, 1980, J Cell Sci., 46: 341-352). The step in the cell cycle that is sensitive to MBC inhibition was ordered to reciprocal shift experiments with respect to the step catalyzed by cdc gene products. Execution of the CDC7 step is required for the initiation of DNA synthesis and for completion of the MBC-sensitive step. Results obtained with mutants (cdc2, 6, 8, 9, and 21) defective in DNA replication and with an inhibitor of DNA replication (hydroxyurea) suggest that some DNA replication required for execution of the MBC-sensitive step but that the completion of replication is not. Of particular interest were mutants (cdc5, 13, 14, 15, 16, 17, and 23) that arrest cell division after DNA replication but before nuclear division since previous experiments had not been able to resolve the pathway of events in this part of the cell cycle. Execution of the CDC17 step was found to be a prerequisite for execution of the MBC- sensitive step; the CDC13, 16 and 23 steps are executed independently of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the CDC14 and 23 steps. These results considerably extend the dependent pathway of events that constitute the cell cycle of S. cerevisiae.     

Genetic and Molecular Analysis of Cdr1/Nim1 in Schizosaccharomyces Pombe

The cdr1 gene in Schizosaccharomyces pombe was identified as a mutation affecting the nutritional responsiveness of the mitotic size control. cdr1 alleles have been further analyzed for genetic interactions with elements of the mitotic control pathway and cloned by plasmid rescue of a conditional lethal cdr1-76 cdc25-22 double mutant. These analyses show that the cdr1 gene is allelic to nim1, a gene identified as a high copy number plasmid suppressor of the mitotic control gene, cdc25. The gene structure for cdr1 differs from the described nim1 gene in the carboxyl-terminal portion of the gene. The published nim1 sequence encoded a product of predicted Mr 45,000, and included 356 amino acids from the amino-terminal region of the gene and 14 amino acids from a noncontiguous carboxyl-terminal fragment. The cdr1 sequence includes an additional 237 amino acids of the contiguous fragment and encodes a product of predicted Mr 67,000. The sequence shows a high level of identity with protein kinases over the amino-terminal catalytic domain, and limited identity with yeast protein kinases SNF1, KIN2 and KIN1 over part of the carboxyl-terminal domain. The effect of overexpression of the full length gene has been examined in various genetic backgrounds. These data show that the full length gene product is required to give a normal cell cycle response to nitrogen starvation. A detailed examination of the genetic interaction of cdr1 mutants with various mutants of mitotic control genes (cdc2, cdc25, wee1, cdc13) demonstrated strong interactions with cdc25, some cdc2 alleles, and with cdc13-117. Overall, the results are interpretable within the framework of the existing model of cdr1/nim1 action in mitotic control, i.e., cdr1 functions upstream of wee1 to relieve mitotic inhibition.     

The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity.     

Mitotic Transmission of Artificial Chromosomes in Cdc Mutants of the Yeast, Saccharomyces Cerevisiae

In the yeast, Saccharomyces cerevisiae, cell division cycle (CDC) genes have been identified whose products are required for the execution of different steps in the cell cycle. In this study, the fidelity of transmission of a 14-kb circular minichromosome and a 155-kb linear chromosome fragment was examined in cell divisions where specific CDC products were temporarily inactivated with either inhibitors, or temperature sensitive mutations in the appropriate CDC gene. All of the cdc mutants previously shown to induce loss of endogenous linear chromosomes also induced loss of a circular minichromosome and a large linear chromosome fragment in our study (either 1:0 or 2:0 loss events). Therefore, the efficient transmission of these artificial chromosomes depends upon the same trans factors that are required for the efficient transmission of endogenous chromosomes. In a subset of cdc mutants (cdc6, cdc7 and cdc16), the rate of minichromosome loss was significantly greater than the rate of loss of the linear chromosome fragment, suggesting that a structural feature of the minichromosome (nucleotide content, length or topology) makes the minichromosome hypersensitive to the level of function of these CDC gene products. In another subset of cdc mutants (cdc7 and cdc17), the relative rate of 1:0 events to 2:0 events differed for the minichromosome and chromosome fragment, suggesting that the type of chromosome loss event observed in these mutants was dependent upon chromosome structure. Finally, we show that 2:0 events for the minichromosome can occur by both a RAD52 dependent and RAD52 independent mechanism. These results are discussed in the context of the molecular functions of the CDC products.     

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.     

Temperature-Sensitive Cdc 7 Mutations of Saccharomyces Cerevisiae Are Suppressed by the Dbf4 Gene, Which Is Required for the G(1)/S Cell Cycle Transition

When present on a multicopy plasmid, a gene from a Saccharomyces cerevisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation. The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle. DBF4 has an open reading frame encoding 695 amino acid residues and the predicted molecular mass of the gene product is 80 kD. The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4. Suppression is mitosis-specific and the sporulation defect of cdc7 mutations is not suppressed by DBF4. Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations. Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations. These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication.     

Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.     

Test for temporal or spatial restrictions in gene product function during the cell division cycle.

The ability of a functional gene to complement a nonfunctional gene may depend upon the intracellular relationship of the two genes. If so, the function of the gene product in question must be limited in time or in space. CDC (cell division cycle) gene products of Saccharomyces cerevisiae control discrete steps in cell division; therefore, they constitute reasonable candidates for genes that function with temporal or spatial restrictions. In an attempt to reveal such restrictions, we compared the ability of a CDC gene to complement a temperature-sensitive cdc gene in diploids where the genes are located within the same nucleus to complementation in heterokaryons where the genes are located in different nuclei. In CDC X cdc matings, complementation was monitored in rare heterokaryons by assaying the production of cdc haploid progeny (cytoductants) at the restrictive temperature. The production of cdc cytoductants indicates that the cdc nucleus was able to complete cell division at the restrictive temperature and implies that the CDC gene product was provided by the other nucleus or by cytoplasm in the heterokaryon. Cytoductants from cdc28 or cdc37 crosses were not efficiently produced, suggesting that these two genes are restricted spatially or temporally in their function. We found that of the cdc mutants tested 33 were complemented; cdc cytoductants were recovered at least as frequently as CDC cytoductants. A particularly interesting example was provided by the CDC4 gene. Mutations in CDC4 were found previously to produce a defect in both cell division and karyogamy. Surprisingly, the cell division defect of cdc4 nuclei is complemented by CDC4 nuclei in a heterokaryon, whereas the karyogamy defect is not.     

CDC17: an essential gene that prevents telomere elongation in yeast

The CDC17 gene product performs an essential stage-specific function during the Saccharomyces cerevisiae cell cycle. When cdc17-1 strains are grown at the maximum permissive temperature, recombination is induced preferentially in the genetic interval of the chromosome closest to the telomere. Telomeres are longer in cdc17 strains than in CDC17 strains at the permissive temperature because of addition of sequence near or in the poly (C1-3A) telomeric DNA and become even longer when cells are propagated at elevated temperatures. The mitotic recombination events require RAD52 function, but telomere growth does not. Long telomeres are maintained for many generations when crossed into a CDC17+ background, suggesting that telomere length is largely conserved during replication. The altered telomere length phenotype of cdc17 mutations is recessive and coreverts and cosegregates with the temperature-sensitive lethal phenotype.     

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.     

Cloning and characterization of the Saccharomyces cerevisiae CDC6 gene.

The yeast cell division cycle gene CDC6 was isolated by complementation of a temperature-sensitive cdc6 mutant with a genomic library. The amino acid sequence of the 48 kDalton CDC6 gene product, as deduced from DNA sequence data, includes the three consensus peptide motifs involved in guanine nucleotide binding and GTPase activity, a target site for cAMP-dependent protein kinase and a carboxy-terminal domain related to metallothionein sequences. A plasmid-encoded CDC6-beta-galactosidase hybrid protein was located at the plasma membrane by indirect immunofluorescence. Disruption experiments indicate that the CDC6 gene product is essential for mitotic growth.     

The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase

The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.