Contribution of a photoreactivable component to the oxygen effect observed in certain rad mutants of Saccharomyces cerevisiae after exposure to high energy electrons

It is shown that a fraction of damage induced by high energy electrons (25 MeV) in certain rad mutants of the yeast Saccharomyces cerevisiae can be photoreactivated. The photoreactivable damage contributes to the lethal effect of this type of irradiation and modifies the oxygen effect. Using photoreactivating light or nigrosin, the amount of photoreactivable damage is reduced and the oxygen enhancement ratio (OER) for yeast mutants increases approximately to the OER found in wild-type cells.     

The formation of photoreactivable damage by direct excitation of DNA in X-irradiated E. coli cells

The role of the direct excitation process in the formation of photoreactivable damage (pyrimidine dimers) in E. coli WP2 hcr-exr- cells has been studied. The pyrimidine dimers were detected by photoreactivation following anoxic irradiation by X-rays (220 kVp). The dose modifying factor (DMF) is 1.28 +/- 0.09. A biophysical model is used for a theoretical examination of the importance of the direct excitation process in the formation of photoreactivable damage and the experimental data are consistent with this model.     

Arsenite-induced lipid peroxidation in Saccharomyces cerevisiae

The ability of sodium arsenite at concentrations of 10(-2), 10(-4), and 10(-6) M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10(-2) and 10(-4) M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10(-6) M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Cadmium-induced oxidative stress in Saccharomyces cerevisiae

The present study was undertaken to determine the effect of cadmium (Cd) on the antioxidant status of the yeast Saccharomyces cerevisiae. S. cerevisiae serves as a good eukaryotic model system for the study of the molecular mechanisms of oxidative stress. We investigated the adaptative response of S. cerevisiae exposed to Cd. Yeast cells could tolerate up to 100 microM Cd and an inhibition in the growth and viability was observed. Exposure of yeast cells to Cd showed an increase in malondialdehyde and glutathione. The activities of catalase, superoxide dismutase and glutathione peroxidase were also high in Cd-exposed cells. The incorporation of Cd led to significant increase in iron, zinc and inversely the calcium, copper levels were reduced. The results suggest that antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumably, these enzymes are essential for counteracting the pro-oxidant effects of Cd.     

Effect of caffeine on ozone-sensitivity in Saccharomyces cerevisiae

Summary The addition of 0.1% caffeine to the plating medium markedly reduced the ozone-survival of the wild-type and the rad1 and rad6 mutants of Saccharomyces cerevisiae, whereas no effect was observed in the rad52 mutant. Since, in S. cerevisiae, caffeine has been reported to interfere with the recombinational repair pathway under the control of the RAD52 gene, these results support previous observations suggesting that this pathway is involved in the repair of ozone-induced DNA damage.     

The effect of sulfite on the yeast Saccharomyces cerevisiae

After a short period of tolerance, living cells of Saccharomyces cerevisiae were irreversibly damaged by low concentrations of sulfite. The length of the period of tolerance and the rate of the damaging effect depended on the concentration on sulfite, pH-value, temperature, the physiological state of the cells, and incubation time.Inhibitors of protein synthesis and mitochondrial ATP synthesis did not alter the deleterious effect of sulfite on living cells. Furthermore, cell damage leading to inhibition of colony formation occured under aerobic as well as under anaerobic conditions.Prior to cell inactivation sulfite induced the formation of respiratory deficient cells.The active agent was shown to be SO₂.     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.     

Membrane damage associated with inositol-less death in Saccharomyces cerevisiae.

The effect of inositol deficiency was studied on fermentation, respiration, and sugar and amino acid transport. It was found that the loss of fermetnation and respiration and sugar transport activity parallel the loss of cell viability. The loss of sugar transport activity is associated with the development of cell membrane damage. It is concluded that the ultimate cause of cell death is cell membrane leakiness.     

Modification of the radiosensitivity of E. coli cells by factors affecting the yield of photoreactivated damage

A study was made of the influence of irradiation conditions on the yield of the photoreactivable damages in radiosensitive mutants of E. coli cells (E. coli WP2). Pyrimidine dimers were shown to occur in exrA- and recA- mutants irradiated under anoxic conditions, the survival of these mutants being modified depending on cell genotype. The processes of direct excitation of the molecules were involved in the formation of the damages observed. It can be assumed that the lesser oxygen effect observed in exrA- and partially in recA- mutants of E. coli WP2 cells is associated with a contribution of the photoreactivable damages to a lethal effect of ionizing radiation.     

Lead toxicity in Saccharomyces cerevisiae

The effect of Pb on Saccharomyces cerevisiae cell structure and function was examined. Membrane integrity was assessed by the release of UV-absorbing compounds and by the intracellular K⁺ efflux. No leakage of UV₂₆₀-absorbing compounds or loss of K⁺ were observed in Pb (until 1,000 μmol/l) treated cells up to 30 min; these results suggest that plasma membrane seems not to be the immediate and primary target of Pb toxicity. The effect of Pb on yeast metabolism was examined using the fluorescent probe FUN-1 and compared with the ability to reproduce, evaluated by colony-forming units counting. The exposition of yeast cells, during 60 min to 1,000 μmol/l Pb, induces a decrease in the ability to process FUN-1 although the cells retain its proliferation capacity. A more prolonged contact time (120 min) of yeast cells with Pb induces a marked (> 50%) loss of yeast cells metabolic activity and replication competence through a mechanism which most likely requires protein synthesis.     

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.     

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.     

Regulation of inositol monophosphatase in Saccharomyces cerevisiae

Inositol monophosphatase is a key enzyme in the de novo biosynthesis of inositol and in the phosphoinositide second-messenger signalling pathway. Inhibition of this enzyme is a proposed mechanism for lithium's pharmacological action in bipolar illness (manic depression). Very little is known about how expression of this enzyme is regulated. Because the yeast Saccharomyces cerevisiae has been shown to be an excellent model system in which to understand the regulation of inositol metabolism, we characterized inositol monophosphatase in this yeast. Lithium inhibited monophosphatase activity in vitro . Growth in the presence of inositol resulted in increased expression of the enzyme in vivo , although inositol had no effect on enzyme activity in vitro . The inositol effect was apparent when cells were grown in glucose but not in glycerol/ethanol. Monophosphatase activity was derepressed as cells entered stationary phase. This effect was apparent only during growth in glucose plus inositol. The results demonstrate that S. cerevisiae monophosphatase is inhibited by lithium and regulated by factors affecting phospholipid biosynthesis.     

Mutagenic effect of freezing on mitochondrial DNA of Saccharomyces cerevisiae

Although suggested in some studies, the mutagenic effect of freezing has not been proved by induction and isolation of mutants. Using a well-defined genetic model, we supply in this communication evidence for the mutagenic effect of freezing on mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae. The cooling for 2 h at +4 degrees C, followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C resulted in induction of respiratory mutations. The immediate freezing in liquid nitrogen was without mutagenic effect. The study of the stepwise procedure showed that the induction of respiratory mutants takes place during the freezing at -10 and -20 degrees C of cells pre-cooled at +4 degrees C. The genetic crosses of freeze-induced mutants evidenced their mitochondrial rho- origin. The freeze-induced rho- mutants are most likely free of simultaneous nuclear mutations. The extracellular presence of cryoprotectants did not prevent the mutagenic effect of freezing while accumulation of cryoprotectors inside cells completely escaped mtDNA from cryodamage. Although the results obtained favor the notion that the mutagenic effect of freezing on yeast mtDNA is due to formation and growth of intracellular ice crystals, other reasons, such as impairment of mtDNA replication or elevated levels of ROS production are discussed as possible explanations of the mutagenic effect of freezing. It is concluded that: (i) freezing can be used as a method for isolation of mitochondrial mutants in S. cerevisiae and (ii) given the substantial development in cryopreservation of cells and tissues, special precautions should be made to avoid mtDNA damage during the cryopreservation procedures.     

Photoreactivation of mutational damage produced by the interaction between DEB and UV in Neurospora

Summary The mutational damage produced by the interaction between DEB-pretreatment and UV-posttreatment in a Neurospora strain, ad-3A (38701) inos (37401), is photoreactivable. It may thus be concluded that the interaction mutants possess characteristics of UV-induced ones.     

The S-phase checkpoint and its regulation in Saccharomyces cerevisiae

Cells are never more vulnerable than during DNA replication, which represents a major moment of potential genetic instability. Genotoxic insults induce many different forms of DNA damage that may interfere with the ability of cells to properly duplicate their genome. Primary damage may in turn undergo structural transformations during DNA replication, thus generating secondary lesions that may be even more dangerous. Cells experiencing replication of damaged DNA or replication blocks activate an S-phase checkpoint response that assures the fidelity and completion of DNA replication before cells enter M-phase. The S-phase checkpoint pathway regulates not only progress through the cell cycle but also DNA repair and DNA replication itself.