Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae

Photoactivated psoralens used in treatment of skin diseases like Psoriasis and Vitiligo cause DNA damage, the repair of which may lead to mutations and thus to higher risk to have skin cancer. The simple eukaryote Saccharomyces cerevisiae was chosen to investigate the cells' genetic endowment with repair mechanisms for this type of DNA damage and to study the genetic consequences of such repair. Genetic studies on yeast mutants sensitive to photoactivated psoralens, named pso mutants, showed their allocation to 10 distinct loci. Cloning and molecular characterization allowed their grouping into three functional classes: (I) the largest group comprises seven PSO genes that are either generally or specifically involved in error-prone DNA repair and thus affect induced mutability and recombination; (II) one PSO gene that represents error-free excision repair, and (III) two PSO genes encoding proteins not influencing DNA repair but physiological processes unrelated to nucleic acid metabolism. Of the seven DNA repair genes involved in induced mutagenesis three PSO loci [PSO1/REV3, PSO8/RAD6, PSO9/MEC3] were allelic to already known repair genes, whereas three, PSO2/SNM1, PSO3/RNR4, and PSO4/PRP19 represent new genes involved in DNA repair and nucleic acid metabolism in S. cerevisiae. Gene PSO2 encodes a protein indispensable for repair of interstrand cross-link (ICL) that are produced in DNA by a variety of bi- and polyfunctional mutagens and that appears to be important for a likewise repair function in humans as well. In silico analysis predicts a putative endonucleolytic activity for Pso2p/Snm1p in removing hairpins generated as repair intermediates. The absence of induced mutation in pso3/rnr4 mutants indicates an important role of this subunit of ribonucleotide reductase (RNR) in regulation of translesion polymerase zeta in error-prone repair. Prp19p/Pso4p influences efficiency of DNA repair via splicing of pre-mRNAs of intron-containing repair genes but also may function in the stability of the nuclear scaffold that might influence DNA repair capacity. The seventh gene, PSO10 which controls an unknown step in induced mutagenesis is not yet cloned. Two genes, PSO6/ERG3 and PSO7/COX11, are responsible for structural elements of the membrane and for a functional respiratory chain (RC), respectively, and their function thus indirectly influences sensitivity to photoactivated psoralens.     

Thermoresistance in Saccharomyces cerevisiae yeasts

Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.     

Intragenic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive mutants of Saccharomyces cerevisiae yeasts

The effect of UV- and gamma-irradiation on the survival and intragenic mitotic recombination (gene conversion) of 5 radiosensitive mutants was studied in comparison with the wild type. The level of spontaneous conversion was similar for RAD, rad2 and rad15, mutations xrs2 and xrs4 increasing and rad54 significantly decreasing it. The frequency of conversion induced by UV-light was greater in rad2, rad15 and xrs2 mutants and lower in xrs4, as compared to RAD. Gamma-irradiation caused induction of gene conversion with an equal frequency in RAD, rad2, rad15. Xrs2 and xrs4 mutations slightly decreased gamma-induced conversion. In rad54 mutant, UV-and gamma-induced conversion was practically absent. In the wild type yeast, a diploid strain is more resistant than a haploid, whereas in rad54 a diploid strain has the same or an increased sensitivity, as compared to a haploid strain (the "inverse ploidy effect"). This effect and also the block of induced mitotic recombination caused by rad54 indicate the presence in the yeast Saccharomyces cerevisiae of repair pathways of UV- and gamma-induced damages acting in diploid cells and realised by recombination. The data obtained as a result of many years' investigation of genetic effects in radiosensitive mutants of yeast are summarised and considered.     

Killer systems of Saccharomyces cerevisiae yeasts

The killer systems of Saccharomyces cerevisiae are a peculiar group of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. Their basic properties are linearity of genome, its fragmentation, resulting in two separately replicating major and minor segments, and the ability to control the synthesis of secretory proteins--mycocins which can kill the taxonomically related strains. Secretion of mycocins also confers immunity to their action. The strains containing killer symbionts are toxigenic and resistant to their own toxins, while those with no killer double-stranded RNA are sensitive to mycocins. The killer systems of Saccharomyces cerevisiae possess some properties relevant to viruses and evidently are evolved during the evolution of infectious viruses. Occurrence of such systems in monocellular eucaryotic organisms is an example of genome complication in the course of putting together the virus-like components. The peculiarities of replication and expression of killer systems and their utilization for the construction of vector molecules are discussed.     

Effect of long-wave ultraviolet light (313 nm) on the survivability and respiration of Candida utilis yeasts

Irradiation of Candida utilis with near-UV (313 nm) was found to inhibit respiration; the degree of the inhibition correlated with the number of killed cells. Irradiation of the yeast mitochondria inhibited the activity of succinate oxidase; the dependence of the inhibition on the dose of near-UV (313 nm) was close to the dose dependence of the photoinhibition of respiration in whole cells. The action spectrum for the inhibition of the activity of succinate oxidase was recorded; it corresponded to the absorption spectrum of ubiquinone isolated from the yeast cells. The data obtained suggest that ubiquinone is a target molecule in the effects of inhibition of respiration and death of yeast cells induced with near-UV (313 nm).     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

Loss of heterozygosity and DNA damage repair in Saccharomyces cerevisiae

Loss of heterozygosity (LOH) of tumor suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Understanding how LOH events arise may provide an opportunity for the prevention or early intervention of cancer development. In an effort to investigate the source of LOH events, we constructed MATalphacan1Delta::LEU2 and MATa CAN1 haploid yeast strains and examined canavanine-resistance mutations in a MATa CAN1/MATalphacan1Delta::LEU2 heterozygote formed by mating UV-irradiated and nonirradiated haploids. An increase in LOH was observed when the irradiated CAN1 haploid was mated with nonirradiated can1Delta::LEU2, while reversed irradiation only marginally increased LOH. In the rad51Delta background, allelic crossover type LOH increased following UV irradiation but not gene conversion. In the rad52Delta background, neither type of LOH increased. The chromosome structure following LOH and the requirement for Rad51 and Rad52 proteins indicated the involvement of gene conversion, allelic crossover and break-induced replication. We argued that LOH events could have occurred during the repair of double-strand breaks on a functional (damaged) but not nonfunctional (undamaged) chromosome through recombination.     

High-pressure CO2 inactivation and induced damage on Saccharomyces cerevisiae evaluated by flow cytometry

The cultivability, integrity and permeabilisation of Saccharomyces cerevisiae in saline solution after high-pressure CO₂ treatment at 36 °C was assessed by using both conventional cultivation-based technique and flow cytometry. Conventional cultivation-based techniques do not allow the exact quantification of integer cells, which can be determined coupling the staining with propidium iodide and SYBR-Green I and the cell quantification by flow cytometry. A significant portion of cells injured by CO₂ treatment is incapable of forming colonies but is still integer and potentially metabolically active. The yeast cell damage was demonstrated to be dependent on the conditions applied. In particular the influence of different operative parameters on integrity and permeabilisation of yeast cells was evaluated: pressure (50–100 bar), treating time (10–20 min) and stirring rate (500–10,000 rpm). After a 20 min treatment at 100 bar, 36 °C and 10,000 rpm more than 95% of cells result with completely permeabilised membrane.     

Acetoin production in Saccharomyces cerevisiae wine yeasts

Abstract One hundred strains of Saccharomyces cerevisiae were examined for the capacity to produce acetoin in synthetic medium and in grape must. The low production of acetoin was found to be the more common pattern in this species. Most strains exhibited a similar distribution in both media, production ranging from non-detectable amounts to 12 mg 1⁻¹. Only four strains produced high quantities of acetoin, up to 29.5 mg l⁻¹ in synthetic medium and up to 194.6 mg l⁻¹ in grape must. This biometric study showed the existence of two phenotypes, "low and high acetoin production", that could be selected for conferring a desirable flavour of the final product.     

Replication in Saccharomyces cerevisiae of plasmid pBR313 carrying DNA from the yeast trpl region.

Plasmid pBR313 carrying a 1.4 kb EcoRI fragment from the yeast TRP1 region (designated pLC544) is capable of transforming yeast trp1 mutants to Trp+ at high frequency (10(3)--10(4) transformants/micrograms DNA). Transformation can be achieved either by using purified plasmid DNA or by fusion of yeast spheroplasts with partially lysed Escherichia coli [pLC544] protoplast preparations. The Trp+ yeast transformants are highly unstable, segregating Trp- cells at frequencies of 0.18 per cell per generation (haploids) and 0.056 per cell per generation (diploids) in media containing tryptophan. Plasmid pLC544 replicates autonomously in the nucleus of yeast cells and segregation of Trp-cells is associated with the complete loss of plasmid sequences. In genetic crosses, pLC544 is randomly assorted during meiosis and is carried unchanged through the mating process into haploid recombinants.     

Genetic differentiation of the sherry yeasts Saccharomyces cerevisiae

Molecular genetic study of the yeast Saccharomyces cerevisiae isolated at various stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from the primary winemaking yeasts according to several physiological and molecular markers. All sherry strains independently of the place and time of their isolation carry a 24-bp deletion in the ITS 1 region of ribosomal DNA, whereas the yeasts of the primary winemaking lack this deletion. Molecular karyotypes of the sherry yeast from different populations were found very similar.     

Role of Dot1 in the response to alkylating DNA damage in Saccharomyces cerevisiae: regulation of DNA damage tolerance by the error-prone polymerases Polzeta/Rev1

Maintenance of genomic integrity relies on a proper response to DNA injuries integrated by the DNA damage checkpoint; histone modifications play an important role in this response. Dot1 methylates lysine 79 of histone H3. In Saccharomyces cerevisiae, Dot1 is required for the meiotic recombination checkpoint as well as for chromatin silencing and the G(1)/S and intra-S DNA damage checkpoints in vegetative cells. Here, we report the analysis of the function of Dot1 in the response to alkylating damage. Unexpectedly, deletion of DOT1 results in increased resistance to the alkylating agent methyl methanesulfonate (MMS). This phenotype is independent of the dot1 silencing defect and does not result from reduced levels of DNA damage. Deletion of DOT1 partially or totally suppresses the MMS sensitivity of various DNA repair mutants (rad52, rad54, yku80, rad1, rad14, apn1, rad5, rad30). However, the rev1 dot1 and rev3 dot1 mutants show enhanced MMS sensitivity and dot1 does not attenuate the MMS sensitivity of rad52 rev3 or rad52 rev1. In addition, Rev3-dependent MMS-induced mutagenesis is increased in dot1 cells. We propose that Dot1 inhibits translesion synthesis (TLS) by Polzeta/Rev1 and that the MMS resistance observed in the dot1 mutant results from the enhanced TLS activity.