Role of the CDC8 gene in the repair of single strand breaks in DNA of the yeast Saccharomyces cerevisiae.

It has been found that the repair of single strand breaks is defective in the DNA replication mutants cdc8-1 and cdc8-3 of Saccharomyces cerevisiae both in permissive (23 degrees C) and restrictive conditions (36 degrees C). In permissive conditions we observed a significant delay in single strand break repair in a diploid strain HB7 (cdc8-1/cdc8-1), as compared with the wild-type strain. Under restrictive conditions no repair was observed, but rather degradation of MMS-damaged DNA occurred. It has been also found that the repair of single strand breaks in yeast is inhibited by cycloheximide but not by hydroxyurea.

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene.

Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation. It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.

Induced recombination and reversion of the CDC8 gene in relation to the cell cycle of yeast.

The induction of mitotic recombination in the CDC8 locus was studied in a diploid strain heteroallelic for cdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strains cdc8-1/cdc8-1 and cdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction of cdc+ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle-G1, S and G2. It was also found that UV-induced gene reversion can occur during the S and G2 stages, but not during the G1 stage of the cell cycle.

Alkaline elution of yeast DNA: a simple method of detection of DNA single-strand breaks.

A method of analysis has been developed to study the induction and repair of single-strand breaks in yeast DNA by alkaline elution from filters. By this technique sphaeroplasts obtained from yeast cells were lysed on the filters and single-strand DNA fragments selectively eluted by alkaline elution. Three different approaches are proposed: (i) pH step alkaline elution from the filters; (ii) alkaline elution from the filters using one selected level of pH; (iii) alkaline elution from the filter in which fractions were collected directly on Whatman 17 glass filter paper, thus simplifying the procedure. We found that the first method was the most sensitive, while the next two were much simpler but less sensitive. On the other hand, these two methods enabled us to express semiquantitatively the degree of DNA damage and repair.

Decreased u.v. mutagenesis in an excision-deficient mutant of yeast.

The u.v.-sensitive rad4 mutant of yeast was found to decrease u.v.-induced reversion in the cdc8 and lys2 loci. Survival curves obtained following u.v. irradiation and u.v.-induced reversion in rad4, cdc8 and cdc8rad4 double mutants show that cdc8 is epistatic to rad4 and belongs to the 'rad3' epistasis group. A study of u.v.-induced reversion in the thermosensitive cdc8 mutant at the restrictive temperature (prevents DNA synthesis) and at the permissive temperature (DNA synthesis takes place) indicates the essential role of DNA replication in u.v.-induced mutagenesis.

Intracellular localization of Aspergillus nidulans ornithine carbamoyltransferase in native host cells and in Saccharomyces cerevisiae cells harbouring its cloned structural gene.

Differential centrifugation of the Aspergillus nidulans cell lysate shows that ornithine carbamoyltransferase (EC 2.1.3.3) appears mainly in the particulate (organellar) fraction. The enzyme was located to the mitochondria by co-sedimentation with cytochrome oxidase in isopycnic density gradient and by cytochemical-electron microscopic means. Arginase (EC 3.5.3.1) and ornithine delta-aminotransferase (E.C. 2.6.1.13) were found to reside in cytosol. The release of ornithine carbamoyltransferase from the organellar fraction by various agents indicates that the enzyme resides in the mitochondrial matrix. In Saccharomyces cerevisiae the plasmid pSAL43, carrying cloned Aspergillus nidulans ornithine carbamoyltransferase gene, directs the synthesis of the enzyme partially associated with yeast mitochondria even though the homologous yeast enzyme is exclusively cytosolic. The implications of these findings are discussed.

The role of respiratory chain in paraquat toxicity in yeast.

Yeast respiratory deficient mutants are resistant to paraquat. Similar resistance is caused by lowering the respiration by physiological mechanisms, as well as by some inhibitors of electron transfer chain of mitochondria. Presented results suggest that the major contribution of mitochondria to overall toxicity of paraquat in yeast is a consequence of very low level of cytochrome P-450, and presumably its presumably its reductase in aerobic yeast cells. In mammalian cells this enzyme is considered as the most important factor involved in paraquat toxicity. Mitochondrial cytochromes seem to be the first targets of damaging effects of paraquat.

Analysis of replication of DEB-alkylated DNA in yeast: bypass replication in a rad3 mutant of Saccharomyces cerevisiae.

We presented indirect evidence that in an excision-deficient rad3 mutant of yeast exposed to diepoxybutane (DEB), DNA synthesis continued past the damaged sites. This bypass replication was confined to the first post-treatment round of replication and was followed by inhibition of DNA synthesis. Analyses by alkaline sucrose gradient sedimentation and by alkaline elution from filters revealed that in mutant cells the first post-treatment round of replication proceeded at a similar rate to that in untreated cells and was not accompanied by strand scission of template DNA. The post-treatment synthesis was presumably of an error-prone type, as the frequency of reversion to ade2-1 prototrophy was increased. In contrast, in the isogenic wild-type strain, the post-treatment incorporation of radioactivity into DNA was slightly reduced and newly replicated DNA fragments were of lower molecular weight than in control cells. There was also some strain scission in template DNA, presumably resulting from excision-repair.

Analysis of yeast DNA by alkaline filter elution.

The method of analysis of DNA in mammalian cells by alkaline elution from filters (Kohn et al. 1974) was adapted for studies on yeast DNA. By this technique spheroplasts obtained from yeast cells are lysed on filters and single-stranded DNA fragments selectively eluted by alkaline solutions. The procedure was applied to monitor the occurrence of replication intermediates and production of DNA single-strand breakage by MMS, and its repair in growth medium.

DNA replication in a diploid strain of Saccharomyces cerevisiae homozygous for the rad6-1 mutation.

The generation time of a diploid strain homozygous for the rad6-1 mutation was 160 min, and the duration of the S phase was 80 min; in the parental heterozygote, these values were 90 and 40 min, respectively. Analysis of DNA sedimentation in an alkaline sucrose gradient revealed that heterozygote high-molecular-weight DNA appeared after 60 min, and homozygote high-molecular weight DNA only after a 100-min pulse.

Abnormalities in cell division induced by diepoxybutane in rad1-1 and rad3 mutants of Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants rad1-1 and rad3 differ from the wild-type and from other UV-sensitive rad mutants in their behaviour after transfer from medium containing 1,2:3,4-diepoxybutane (DEB) to DEB-free medium. In both mutants several post-treatment cell cycles proceed in the absence of cell wall separation, resulting in the formation of multicellular chains or aggregates. In this study, electron and light microscopy revealed that at least one post-treatment budding cycle is accompanied by nuclear division while subsequent cell cycles can proceed in the absence of regular nuclear cycles. At low percentage survival levels, the first post-treatment budding cycle was not delayed and was accompanied by significant incorporation of radioactivity into DNA and protein. In contrast, subsequent cell cycles were found to be accompanied by only protein synthesis and not DNA synthesis. The wild-type strain, unlike the mutants, responded to DEB treatment by a dose-dependent lag in the onset of macromolecular synthesis and cell proliferation, and after prolonged incubation in mutagen-free medium the culture consisted of single budded and unbudded cells.

cdc and prt Mutants of Saccharomyces cerevisiae with increased sensitivity to diepoxybutane and ultraviolet.

The sensitivity to UV and DEB of nine temperature-sensitive mutants was investigated. All prt and four cdc mutants (cdc4, cdc7, cdc8, cdc28) showed a higher level of sensitivity to UV than the wild type strain. Three mutants cdc7, cdc9 and prt1 are more sensitive than the wild type to DEB, but only two cdc7 and prt1 are sensitive to UV and DEB. Treatment with restrictive temperature increases the sensitivity after UV treatment of cdc9, cdc21, prt1, prt3 and after DEB treatment of cdc28 and all prt mutants.

Energy requirement for liquid holding recovery from UV- and DEB-induced damage in rad mutants of Saccharomyces cerevisiae.

Respiratory deficient (rho o) strains respond to liquid holding in buffer alone with a sharp decrease of cell survival. In buffer with 0.02% glucose rho o strains behave similarly as rho+ strains. After UV or DEB inactivation rho o strains derived from rho+ rad mutants capable of LHR in buffer alone, manifest this capacity in glucose buffer. In some respiratory proficient rad mutants LHR is enhanced or induced by holding in glucose buffer, e.g. DEB inactivated rad3, rad6 and rad14. In such mutants the number of mitochondrial profiles was found to be 3 to 4 times lower than in the wild-type RAD or in rad mutants with LHR insensitive to glucose. This suggests that mutants with glucose-enhanced LHR are defective in energy metabolism and require exogenous energy supply for initiation of liquid holding repair capacity. In UV-irradiated JG-1 (rad1-1), rad3 and rad11 as well as in DEB-inactivated rad7, rad11, rad19 and rad20 the inability of LHR is a constitutive phenomenon and cannot be overcome by exogenous energy supply.

Relation between liquid-holding recovery, DNA repair, and mitotic recombination in the rad3 mutant of Saccharomyces cerevisiae after treatment with diepoxybutane (DEB).

The rad3 mutant is characterized by a high level of liquid-holding recovery after DEB treatment. The recovery is abolished when the treated cells are postincubated in growth medium, but the effect can be cancelled by suppression of DNA and protein synthesis by specific inhibitors. Alkaline sucrose gradient sedimentation revealed that DEB induces single strand breaks in DNA which are not repaired during post-treatment incubation in growth medium or during LH. Effective repair takes place only when LH is followed by incubation in growth medium. Split-dose treatment applied to test the possible inducibility of repair by LH did not confirm this presumption. In a diploid homozygous for rad3 mutation, DEB induces mitotic inter- and intragenic recombination with very high frequency. Liquid-holding recovery (LHR) was found to be accompanied by an increase in molecular weight of DNA and by a sharp decrease in the frequency of mitotic recombination. The data suggest that recombination events are not involved in LHR pathway.

Comparison of sensitivity and liquid holding recovery in rad mutants of Saccharomyces cerevisiae inactivated by UV and DEB.

Twenty one UV-sensitive rad mutants were tested for their sensitivity towards DEB. All mutants were more sensitive to this treatment than the wild type. Seven mutants were classified as supersensitive to DEB (rad 1-1, 2,3, 6, 15 and 18-2), while only rad2 and rad3 can be classified as supersensitive to UV. For all mutants ability for liquid holding recovery (LHR) after UV and DEB was compared. Mutants rad 1-1, 3, 5, 6, 9 and 11 differ in their response to LH after the two treatments. Survival of rad1-1 and rad3 increases signficantly during LH after DEB but not after UV exposure. In contrast rad5, 6, 11, and 22 show marked LHR after UV but no increase of survival after DEB treatment.

Compartmentation of the amino acid pool in Saccharomycopsis lipolytica.

1. The size of the free amino acid pool in S. lipolytica varies from 250 to 350 micronmoles/g dry wt., and it accounts for about 10% of the total amino acid content; 80-90% of free amino acids, including methionine, is compartmentized in vacuole. S-Adenosylmethionine (SAM) occurs in equal proportions in vacuole and cytoplasm while aspartate and glutamate are mainly cytosolic. 2. The bulk of the methionine overproduced in the ethionine-resistant mutant Etr-13, and of the exogenous methionine derived from the methionine-enriched medium, is stored in vacuole. The amount and distribution of SAM is not affected. 3. Overloading with endogenous methionine results in a significant increase in the total cytosolic amino acid pool and is associated with the increased concentration of arginine, glutamine and lysin; on overloading with exogenous methionine, only lysine is elevated.

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.