Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

The Role of S. CEREVISIAE Cell Division Cycle Genes in Nuclear Fusion

Forty temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae were examined for their ability to complete nuclear fusion during conjugation in crosses to a CDC parent strain at the restrictive temperature. Most of the cdc mutant alleles behaved as the CDC parent strain from which they were derived, in that zygotes produced predominantly diploid progeny with only a small fraction of zygotes giving rise to haploid progeny (cytoductants) that signalled a failure in nuclear fusion. However, cdc4 mutants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy defect. For all four mutants, the karyogamy defect and the cell cycle defect cosegregated, suggesting that both defects resulted from a single lesion for each of these cdc mutants. Therefore, the cdc 4, 28, 34 and 37 gene products are required in both cell division and karyogamy.     

Control of Saccharomyces cerevisiae 2microN DNA replication by cell division cycle genes that control nuclear DNA replication.

The replication of the 2 μm DNA of Saccharomyces cerevisiae has been examined in cell division cycle (cdc) mutants. The 2 μm DNA does not replicate at the restrictive temperature in cells bearing the cdc28, cdc4, and cdc7 mutations which prevent passage of cells from the G1 phase into S phase. Plasmid replication also is prevented in a mating-type cells by α factor, a mating hormone which prevents cells from completing an event early in G1 phase. The 2 μm DNA ceases replication at 36 °C in a mutant harboring the cdc8 mutation, a defect in the elongation reactions of nuclear DNA replication. Plasmid replication continues at the restrictive temperature for approximately one generation in a cdc13 mutant defective in nuclear division. These results show that 2 μm DNA replication is controlled by the same genes that control the initiation and completion of nuclear DNA replication.     

Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-Methylsalicylic acid synthase gene by heterologous expression

The geneCAL1 (also known asCDC43) ofSaccharomyces cerevisiae encodes theβ subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded bycall-1, andcdc43-2 tocdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of thecall/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. Thecall-1 mutation, located most proximal to the C-terminus of the protein, differs from the othercdc43 mutations in several respects. An increase in soluble Rholp was observed in thecall-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype ofcall-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious incall-1 cells, but not in othercdc43 mutants or the wild-type strains. Thecdc43-5 mutant cells accumulate Cdc42p in soluble pools andcdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among thecall/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.     

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.     

The effect of the cdc9 mutation on premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

A diploid homozygous for cdc9, a conditional mutation defective in DNA ligase [2], has been used to investigate the role of this enzyme in premeiotic DNA synthesis. The cdc9 ligase has the same effect on premeiotic as on mitotic DNA synthesis and at the restrictive temperature the newly synthesized DNA is recovered in small fragments. A difference has been observed, however, between meiotic and mitotic cells, namely in their ability to join together these fragments on return to the permissive temperature. In mitotic cells this can be readly demonstrated within 50 min, whereas in contrast little joining was detected in meiotic cells, even after 2 h at the permissive temperature.     

Separation of phenotypes in mutant alleles of the Schizosaccharomyces pombe cell-cycle checkpoint gene rad1+.

The Schizosaccharomyces pombe rad1+ gene is involved in the G2 DNA damage cell-cycle checkpoint and in coupling mitosis to completed DNA replication. It is also required for viability when the cdc17 (DNA ligase) or wee1 proteins are inactivated. We have introduced mutations into the coding regions of rad1+ by site-directed mutagenesis. The effects of these mutations on the DNA damage and DNA replication checkpoints have been analyzed, as well as their associated phenotypes in a cdc17-K42 or a wee1-50 background. For all alleles, the resistance to radiation or hydroxyurea correlates well with the degree of functioning of checkpoint pathways activated by these treatments. One mutation, rad1-S3, completely abolishes the DNA replication checkpoint while partially retaining the DNA damage checkpoint. As single mutants, the rad1-S1, rad1-S2, rad1-S5, and rad1-S6 alleles have a wild-type phenotype with respect to radiation sensitivity and checkpoint functions; however, like the rad1 null allele, the rad1-S1 and rad1-S2 alleles exhibit synthetic lethality at the restrictive temperature with the cdc17-K42 or the wee1-50 mutation. The rad1-S5 and rad1-S6 alleles allow growth at higher temperatures in a cdc17-K42 or wee1-50 background than does wild-type rad1+, and thus behave like "superalleles." In most cases both chromosomal and multi-copy episomal mutant alleles have been investigated, and the agreement between these two states is very good. We provide evidence that the functions of rad1 can be dissociated into three groups by specific mutations. Models for the action of these rad1 alleles are discussed. In addition, a putative negative regulatory domain of rad1 is identified.     

Sequential function of gene products relative to DNA synthesis in the yeast cell cycle

An experimental rationale for deciphering the relative dependence of steps in a developmental pathway (Jarvik & Botstein, 1973; Hereford & Hartwell, 1974) has been employed to determine the relationship between the hydroxyurea-sensitive step and various temperature-sensitive steps in the cell cycle of Saccharomyces cerevisiae. Since hydroxyurea inhibits DNA replication in yeast (Slater, 1973), the data identify gene products upon whose function DNA replication is dependent (cdc 4, 6, 7, 2, 8, 21) and gene products whose function or synthesis requires DNA replication (cdc 2, 8, 21, 9, 13, 16, 23, 5, 15). Other gene products (cdc 3, 11, 24) function independent of DNA replication. These results suggest that the events of the cell cycle occur in a proscribed order because many of the gene products that mediate these events arc restricted to a prescribed sequence of function.Mutations in two genes (cdc 2 and 6) result in cells that remain sensitive to hydroxyurea after an incubation at the restrictive temperature, despite the fact that both mutants incorporate radioactive precursors into DNA at the restrictive temperature (Hartwell, 1973). It is suggested that cdc 6 specifies a function that is necessary for the proper initiation of DNA replication, and cdc 2 a function that is necessary for correct DNA elongation, and that in the absence of either of these functions the DNA that is made is either faulty or incomplete.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

The cdc9 ligase joins completed replicons in baker''s yeast

Summary The drug hydroxyurea has been found to affect the conditional DNA ligase mutant cdc9 in the same way as a wild type. Specific concentrations inhibit the joining of completed replicons leading to a substantial accumulation of these molecules. Upon removal of hydroxyurea and further incubation of cdc9 cells at the permissive temperature the replicons joined together, while in sharp contrast at the restrictive temperature no such joining occurred. However, a revertant of cdc9 able to grow at the restrictive temperature was also able to join replicons under these conditions, so the cdc9 ligase must be responsible for the assembly of completed replicons.     

Arrest of the mitotic cell cycle and of meiosis in Saccharomyces cerevisiae by MMS

Summary Methyl methane sulphonate (MMS) was found to arrest mitotic cells at a specific stage in the cell cycle. Reciprocal double shift experiments involving MMS and temperature shifts in several temperature-sensitive cell-cycle (cdc) mutants have located the MMS-sensitive stage after the cdc7 and cdc8 temperature-sensitive stages and before the cdc13, cdc5 and cdc14 stages. An interdependent relationship was found between the arrests caused by MMS, cdc40 and hydroxyurea. Marked increases in mitotic recombination were induced by MMS, both in diploid and haploid strains. Meiosis is arrested by MMS at a very early stage, before DNA replication.     

Ethanol-hypersensitive and ethanol-dependent cdc − mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^? mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^? mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^? phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Genetic control of the cell division cycle in yeast : II. Genes controlling DNA replication and its initiation

Temperature-sensitive mutations occurring in two unlinked complementation groups, cdc4 and cdc8, are recessive and result in a defect in DNA replication at the restrictive temperature. Results obtained with synchronous cultures suggest that cdc4 functions in the initiation of DNA replication and cdc8 functions in the propagation of DNA replication.     

The possible functional significance of phosphatidylinositol in G1 arrest of Saccharomyces cerevisiae

Individual phospholipids were assayed in exponentially growing and G₁-arrested temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that cdc28 cells which are known to arrest at ‘start’ when shifted to their non-permissive temperature, resulted in a 40% decrease in phosphatidylinositol (PI) level while the phosphatidylserine (PS) content was doubled in these cells. The reduced level of PI was restored in cdc4 and cdc7 mutants which are known to arrest past the ‘start’. The increase in PS level in cdc8 mutant which was probably to compensate the intrinsic charging of membrane environment, was also reduced in cdc4 and cdc7 mutants. Our results demonstrate that PI may play a role in yeast cell division and growth that the abnormalities of cdc28 could also be related to PI decrease.     

Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-Methylsalicylic acid synthase gene by heterologous expression

The geneCAL1 (also known asCDC43) ofSaccharomyces cerevisiae encodes the subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded bycall-1, andcdc43-2 tocdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of thecall/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. Thecall-1 mutation, located most proximal to the C-terminus of the protein, differs from the othercdc43 mutations in several respects. An increase in soluble Rholp was observed in thecall-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype ofcall-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious incall-1 cells, but not in othercdc43 mutants or the wild-type strains. Thecdc43-5 mutant cells accumulate Cdc42p in soluble pools andcdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among thecall/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe

Summary A total of 59 new temperature sensitive cdc mutants are described which grow normally at 25°C but become blocked at DNA replication or mitosis when incubated at 36°C. Thirtynine of the mutants are altered in cdc genes which have been identified previously. The remaining 20 mutants define 10 new cdc genes. These have been characterised physiologically, and 6 of the genes (cdc 17, 20, 21, 22, 23, 24) were found to be required for DNA replication, 2 for mitosis (cdc 27, 28), and 2 (cdc 18, 19), could not be unambigously assigned to either DNA replication or mitosis but were definitely required for one or the other.Three genes, the previously identified cdc 10, and cdc 20, 22 are likely to be required for the initiation of DNA replication. Mutants in two genes, cdc 17, 24 undergo bulk DNA synthesis at 36°C, but this DNA is defective. In the case of cdc 17 the defect is in the ligation of Okazaki fragments. cdc 23 is required for bulk DNA synthesis, whilst cdc 21 may possibly be required for the initiation of a particular sub-set of replicons.A previously isolated mutant cdc 13.117 is also further described. This mutant becomes blocked in the middle of mitosis with apparently condensed chromosomes.     

A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25°?C and temperature-sensitive for growth above 33°?C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology.     

Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.