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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Ctf4p facilitates Mcm10p to promote DNA replication in budding yeast

Ctf4p (chromosome transmission fidelity) has been reported to function in DNA metabolism and sister chromatid cohesion in Saccharomyces cerevisiae. In this study, a ctf4^S143F mutant was isolated from a yeast genetic screen to identify replication-initiation proteins. The ctf4^S143F mutant exhibits plasmid maintenance defects which can be suppressed by the addition of multiple origins to the plasmid, like other known replication-initiation mutants. We show that both ctf4^S143F and ctf4Δ strains have defects in S phase entry and S phase progression at the restrictive temperature of 38 °C. Ctf4p localizes in the nucleus throughout the cell cycle but only starts to bind chromatin at the G1/S transition and then disassociates from chromatin after DNA replication. Furthermore, Ctf4p interacts with Mcm10p physically and genetically, and the chromatin association of Ctf4p depends on Mcm10p. Finally, deletion of CTF4 destabilizes Mcm10p and Pol α in both mcm10-1 and MCM10 cells. These data indicate that Ctf4p facilitates Mcm10p to promote the DNA replication.     

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.     

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.     

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.     

Controls over the timing of DNA replication during the cell cycle of fission yeast

The controls acting over the timing of DNA replication (S) during the cell cycle have been investigated in the fission yeast Schizosaccharomyces pombe. The cell size at which DNA replication takes place has been determined in a number of experimental situations such as growth of nitrogen-starved cells, spore germination and synchronous culture of wee mutant and wild-type strains. It is shown that in wee mutant strains and in wild type grown under conditions in which the cells are small, DNA replication takes place in cells of the same size. This suggests that there is a minimum cell size beneath which the cell cannot initiate DNA replication and it is this control which determines the timing of S during the cell cycle of the wee mutant. Fast growing wild-type cells are too large for this size control to be expressed. In these cells the timing of S may be controlled by the completion of the previous nuclear division coupled with a requirement for a minimum period in G1. Thus in S. pombe there are two different controls over the timing of S, either of which can be operative depending upon the size of the cell at cell division. It is suggested that these two controls may form a useful conceptual framework for considering the timing control over S in mammalian cells.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB^A116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB^A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB^A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.     

LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components

After two converging DNA replication forks meet, active replisomes are disassembled and unloaded from chromatin. A key process in replisome disassembly is the unloading of CMG helicases (CDC45–MCM–GINS), which is initiated in Caenorhabditis elegans and Xenopus laevis by the E3 ubiquitin ligase CRL2^LRR1. Here, we show that human cells lacking LRR1 fail to unload CMG helicases and accumulate increasing amounts of chromatin-bound replisome components as cells progress through S phase. Markedly, we demonstrate that the failure to disassemble replisomes reduces the rate of DNA replication increasingly throughout S phase by sequestering rate-limiting replisome components on chromatin and blocking their recycling. Continued binding of CMG helicases to chromatin during G2 phase blocks mitosis by activating an ATR-mediated G2/M checkpoint. Finally, we provide evidence that LRR1 is an essential gene for human cell division, suggesting that CRL2^LRR1 enzyme activity is required for the proliferation of cancer cells and is thus a potential target for cancer therapy.