Temperature-sensitive lethal mutants in the structural gene for dna ligase in the yeast schizosaccharomyces pombe

cdc 17-K42 was isolated as a temperature-sensitive cdc^? mutant of the fission yeast Schizosaccharomyces pombe after nitrosoguanidine mutagenesis. The temperature-sensitive phenotype segregrates 2:2 in tetrad analyses, and it is recessive to the wild-type allele. The pattern of cell division in this mutant on temperature shift implies that its defective function is usually completed by the end of S phase. Cells of cdc 17-K42 enter S phase and undergo a complete round of DNA synthesis at the restrictive temperature, but mitosis does not follow. The nascent DNA accumulated at the restrictive temperature is exclusively composed of short (Okazaki) fragments. After a 20 min pulse label, the main peak of labeled DNA is from 70–450 nucleotides long. DNA ligase assays, involving the formation of covalently closed λ DNA circles, show that the mutant has low levels of DNA ligase activity (<20%) when assayed at the permissive temperature and none detectable when assayed at the restrictive temperature. This implies that the cdc 17 locus codes for the structural gene for DNA ligase. cdc 17-K42 also has a temperature-enhanced ultraviolet sensitivity, suggesting that the same enzyme is involved in DNA repair. Two other independent mutant alleles in the same gene have also been isolated (M75 and L16). They share many of the above properties.     

Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae

The meiotic effects of several cell division cycle (cdc) mutations of Saccharomyces cerevisiae have been investigated by electron microscopy and by genetic and biochemical methods. Diploid strains homozygous for cdc mutations known to confer defects on vegetative DNA synthesis were subjected to restrictive conditions during meiosis. Electron microscopy revealed that all four mutants were conditionally arrested in meiosis after duplication of the spindle pole bodies but before spindle formation for the first meiotic division. None of these mutants became committed to recombination or contained synaptonemal complex at the meiotic arrest. — The mutants differed in their ability to undergo premeiotic DNA synthesis under restrictive conditions. Both cdc8 and cdc21, which are defective in the propagation of vegetative DNA synthesis, also failed to undergo premeiotic DNA synthesis. The arrest of these mutants at the stage before meiosis I spindle formation could be attributed to the failure of DNA synthesis because inhibition of synthesis by hydroxyurea also caused arrest at this stage. — Premeiotic DNA synthesis occurred before the arrest of cdc7, which is defective in the initiation of vegetative DNA synthesis, and of cdc2, which synthesizes vegetative DNA but does so defectively. The meiotic arrest of cdc7 homozygotes was partially reversible. Even if further semiconservative DNA replication was inhibited by the addition of hydroxyurea, released cells rapidly underwent commitment to recombination and formation of synaptonemal complexes. The cdc7 homozygote is therefore reversibly arrested in meiosis after DNA replication, whereas vegetative cultures have previously been shown to be defective only in the initiation of DNA synthesis.     

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.     

Commitment of Mitotic Cells to Meiosis during the G2 Phase of Premeiosis

In the developing anther, archesporial cells that proliferateby mitotic division are converted into meiotic cells duringthe premeiotic interphase. Experiments with explanted microsporocytesof Lilium and Trillium were made to obtain evidence for theconversion of mitotic to meiotic cells during the premeioticperiod. Explanted premeiotic cells were cultured through thedivision cycle at relatively high division frequencies and showeda variety of division types with respect to chromosomal events.The type of division depended on the premeiotic stage at whichthe cells were explanted. Cells in the G₁, S and early G² phasesunderwent mitotic division and formed a diad or binucleate monad.Cells explanted at the late G₂ phase were cultured throughoutthe normal meiotic cycle, which resulted in typical tetrad configuration. In microsporocytes explanted during the main part of the G₂interval, centromere behavior was meiotic, but chromosome pairingand chiasma formation were disturbed. Thus, she G₂ intervalwas shown to be critical for the commitment of mitotic cellsto meiotic division. Detailed analysis showed that the intracellularchanges that commit the cells to meiosis begin shortly aftercompletion of premeiotic DNA synthesis and that these changesare progressive and cumulative. (Received February 2, 1982; Accepted May 24, 1982)     

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.     

A meiosis-specific protein kinase, Ime2, is required for the correct timing of DNA replication and for spore formation in yeast meiosis

 In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase α-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. At late meiotic times, ime2Δ diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase α is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase α are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation. Received: 20 February 1996 / Accepted: 7 June 1996     

Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis

Four steps are known to be required for the initiation of DNA synthesis in Saccharomyces cerevisiae. Three of these are mediated by the products of genes cdc 4, 7, and 28 and the fourth is identified by the inhibition exerted on haploid α cells by the mating pheromone, α factor. These four steps have been ordered by a combination of two methods and found to be:

initiation of DNA synthesis The two sequencing methods are described in detail. Experiments involving the shift of mutant cells from the restrictive to the permissive temperature in the presence of cycloheximide demonstrated that the protein synthesis requirement for yeast DNA replication can be completed before the cdc 7-mediated step.     

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.     

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.     

Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis

Temperature-sensitive mutations in one gene (cdc1) of Saccharomyces cerevisiae confer a defect in bud emergence. Asynchronous cultures of cells defective in cdc1 collect uniformly as unbudded cells (or cells with very tiny buds) following a shift from the permissive to the restrictive temperature. Studies with synchronous cultures demonstrate that the thermolabile product of cdc1 completes its function (the execution point) for bud emergence at the time of bud emergence (0.2 fractions of a cell cycle). When this function is not completed at the restrictive temperature. cells complete DNA replication but do not undergo nuclear division.     

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.     

Initiation of meiosis in cell cycle initiation mutants of Saccharomyces cerevisiae

Control of the initiation of meiosis in yeast was examined in diploids homozygous for one of four different temperature-sensitive mutations that affect “start” of the mitotic cell cycle. Two of the mutations, cdc28 and tra3, bring about deficiencies in the initiation of meiosis, while cdc25 and cdc35 do not prevent initiation of normal meiosis at both permissive and restrictive temperatures. Moreover, diploids homozygous for the latter two mutations are capable of initiating meiosis in rich growth media upon transfer to the high, non-permissive temperature. This unique feature contrasts with the behavior of other yeast strains which require a starvation sporulation medium for initiation of meiosis. It is suggested that the initiation of meiosis includes functions that are shared with “start” of the mitotic cell cycle, as well as functions related to the choice between the two processes. Meiosis in vegetative media at the restrictive temperature (in cdc25 or cdc35 homozygotes) may be important for the study of chemical and physiological phenomena resulting from the meiotic process and not from adaptation to the sporulation medium.     

Isolation, characterisation and molecular cloning of new mutant alleles of the fission yeast p34cdc2+ protein kinase gene: identification of temperature-sensitive G2-arresting alleles

Summary The protein serine-threonine kinase p34 ^cdc2+ plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. p34 ^cdc2+ function is required both for the initiation of DNA replication and for entry into mitosis, and is also required for the initiation of the second meiotic nuclear division. Recent extensive analysis of p34 ^cdc2+ homologue proteins in higher eukaryotes has demonstrated that p34 ^cdc2+ function is likely to be conserved in all eukaryotic cells. Here we report the isolation and characterisation of five new temperature-sensitive alleles of the cdc ²⁺ gene. All five have been cloned and sequenced, together with the meiotically defective cdc2-N22 allele, bringing the total of p34 ^cdc2+ mutants cloned in this and previous reports to seventeen. The five temperature-sensitive alleles define four separate mutations within the p34 ^cdc2+ protein sequence, two of which give rise to cell cycle arrest in G₂ only, when shifted to the restrictive temperature. The nature of the mutation in each protein is described and possible implications for the structure and function of p34 ^cdc2+ discussed.     

The Cyclin-dependent Kinase Inhibitor Dacapo Promotes Genomic Stability during Premeiotic S Phase

The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. Here, we identify the p21^Cip1/p27^Kip1/p57^Kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap^−/− females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, we find that dap^−/− ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap^−/− DNA damage phenotype. Importantly, the DNA damage observed in dap^−/− ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, our data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. Finally, we report that dap^−/− ovarian cysts frequently undergo an extramitotic division before meiotic entry, indicating that Dap influences the timing of the mitotic/meiotic transition.     

Mitotic gene conversion can be as important as meiotic conversion in driving genetic variability in plants and other species without early germline segregation

In contrast to common meiotic gene conversion, mitotic gene conversion, because it is so rare, is often ignored as a process influencing allelic diversity. We show that if there is a large enough number of premeiotic cell divisions, as seen in many organisms without early germline sequestration, such as plants, this is an unsafe position. From examination of 1.1 million rice plants, we determined that the rate of mitotic gene conversion events, per mitosis, is 2 orders of magnitude lower than the meiotic rate. However, owing to the large number of mitoses between zygote and gamete and because of long mitotic tract lengths, meiotic and mitotic gene conversion can be of approximately equivalent importance in terms of numbers of markers converted from zygote to gamete. This holds even if we assume a low number of premeiotic cell divisions (approximately 40) as witnessed in Arabidopsis. A low mitotic rate associated with long tracts is also seen in yeast, suggesting generality of results. For species with many mitoses between each meiotic event, mitotic gene conversion should not be overlooked.

Gene conversion associated with meiosis has long been a focus of attention in population genomics, but mitotic conversion has been relatively overlooked as it was thought to be rare. Analysis in plants suggests that this could be a mistake; long tract lengths and multiple mitoses in species lacking germline sequestration suggest that mitotic conversion, although rare per mitosis, should not be ignored.     

Effects of the Mitotic Cell-Cycle Mutation cdc4 on Yeast Meiosis

The mitotic cell-cycle mutation cdc4 has been reported to block the initiation of nuclear DNA replication and the separation of spindle plaques after their replication. Meiosis in cdc4/cdc4 diploids is normal at the permissive temperature (25 degrees) and is arrested at the first division (one-nucleus stage) at the restrictive temperature (34 degrees or 36 degrees). Arrested cells at 34 degrees show a high degree of commitment to recombination (at least 50% of the controls) but no haploidization, while cells arrested at 36 degrees are not committed to recombination. Meiotic cells arrested at 34 degrees show a delayed and reduced synthesis of DNA (at most 40% of the control), at least half of which is probably mitochondrial. It is suggested that recombination commitment does not depend on the completion of nuclear premeiotic DNA replication in sporulation medium.--Transfer of cdc4/cdc4 cells to the restrictive temperature at the onset of sporulation produces a uniform phenotype of arrest at a 1-nucleus morphology. On the other hand, shifts of the meiotic cells to the restrictive temperature at later times produce two additional phenotypes of arrest, thus suggesting that the function of cdc4 is required at several points in meiosis (at least at three different times).     

Coprinus cinereus DNA ligase I during meiotic development

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.