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.     

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.     

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.     

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.     

Replicating circular DNA molecules in yeast

The yeast Saccharomyces cerevisiae contains a class of small circular DNA molecules, approximately 2 μm in contour length (Sinclair et al., 1967). In this report, it is shown that these molecules replicate as double-branched circles, similar to those observed during replication of the bacteriophage λ and Escherichia coli chromosomes. A normal rate of replication of these DNA circles requires the function of a nuclear gene, cdc 8.     

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 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.     

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.     

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.     

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.     

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.     

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.     

<Emphasis Type="Italic">E. coli</Emphasis> DNA Polymerase I and other Host Functions participate in T4 DNA Replication and Recombination

The recognition of bacterial functions involved in DNA metabolism of bacteriophage T4 might reveal interactions between different enzymes during DNA replication and recombination. To detect such functions we have studied the replication of complete and incomplete T4 chromosomes in various mutant strains of Escherichia coli that are defective in their own DNA metabolism. We found that several E. coli functions can substitute for phage functions in T4 replication and recombination and will discuss here the role of the E. coli pol A gene which codes for DNA polymerase I^1–4 and of the dna B and E genes^3,5.     

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.     

Cold-Sensitive Cell-Division-Cycle Mutants of Yeast: Isolation, Properties, and Pseudoreversion Studies

We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.—Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37°. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.     

Drosophila homologue of the Rothmund-Thomson syndrome gene: essential function in DNA replication during development

Members of the RecQ family play critical roles in maintaining genome integrity. Mutations in human RecQL4 cause a rare genetic disorder, Rothmund-Thomson syndrome. Transgenic mice experiments showed that the RecQ4 null mutant causes embryonic lethality. Although biochemical evidence suggests that the Xenopus RecQ4 is required for the initiation of DNA replication in the oocyte extract, its biological functions during development remain to be elucidated. We present here our results in establishing the use of Drosophila as a model system to probe RecQ4 functions. Immunofluorescence experiments monitoring the cellular distribution of RecQ4 demonstrated that RecQ4 expression peaks during S phase, and RecQ4 is expressed only in tissues active in DNA replication, but not in quiescent cells. We have isolated Drosophila RecQ4 hypomorphic mutants, recq^EP and recq4²³, which specifically reduce chorion gene amplification of follicle cells by 4-5 fold, resulting in thin and fragile eggshells, and female sterility. Quantitative analysis on amplification defects over a 14-kb domain in chorion gene cluster suggests that RecQ4 may have a specific function at or near the origin of replication. A null allele recq4¹⁹ causes a failure in cell proliferation, decrease in DNA replication, chromosomal fragmentation, and lethality at the stage of first instar larvae. The mosaic analysis indicates that cell clones with homozygous recq4¹⁹ fail to proliferate. These results indicate that RecQ4 is essential for viability and fertility, and is required for most aspects of DNA replication during development.     

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.     

Cell cycle inhibition of yeast spheroplasts

Summary Osmotically stabilized yeast spheroplasts are capable of extensive DNA synthesis. Although the rate of DNA synthesis in spheroplasts is approximately one-third that of intact cells, the relative amounts of nuclear and mitochondrial DNA synthesized by spheroplasts is very similar to the relative amounts synthesized by intact cells. Furthermore, nuclear but not mitochondrial DNA synthesis is inhibited in MATa spheroplasts by the application of the yeast mating pheromone, -factor. Similarly, DNA synthesis is reversibly temperature-sensitive in spheroplasts created from cdc7 and cdc8 mutant cells.