The response to chemical mutagens of the individual haploid and homoallelic diploid UV-sensitive mutants of the rad 3 locus of Saccharomyces cerevisiae

Summary Mutant cultures of yeast defective at the generad 3 show increased sensitivity to the lethal effects of UV light. The order of UV sensitivity shown by haploid and homoallelic diploid cultures carrying the variousrad 3 alleles was duplicated by their sensitivity to the action of nitrous acid. In contrast, after treatment with the alkylating agents ethyl-methane sulphonate and methylmethane sulphonate therad 3 cultures showed only small differences in sensitivity compared with the wild-typeRAD culture. These small differences in sensitivity appear to result from variation in the metabolic condition of the cultures when treated with alkylating agents.The results indicate that the product of therad 3 gene in yeast is involved in the repair of UV induced pyrimidine dimers and deaminated bases produced by nitrous acid but does not participate in the repair of single strand DNA breaks produced by alkylating agents.     

The genetic effects of liquid holding recovery in ultra violet light sensitive mutants of yeast

Summary The effect of liquid holding treatment after UV exposure has been determined in diploid yeast cultures carrying genes for UV sensitivity in both the homozygous and heterozygous condition. In all cultures liquid holding treatment produced increases in cell viability, but in cultures carrying a UVS gene this was not correlated with the increase in the frequency of intragenic recombinants characteristic of cultures wild type for UV sensitivity, but with a significant decrease in recombinant frequency. The data presented indicates that the reduction in recombinant frequency was not correlated with a reduction in the total number of recombinants after liquid holding treatment but was due to a selective increase in the frequency of non-recombinant cells.     

The modification of induced genetic change in yeast by an amino acid analogue

Summary Treatment of diploid yeast cultures with the amino acid analogue, para-fluorophenylalanine (PFPA), at concentrations which caused inhibition of growth, resulted in up to 5 fold increases in the frequency of mitotic gene conversion at two different heteroallelic loci. With haploid yeast cultures, growth in PFPA increased the rate of forward mutation to canavanine resistance by at least 2 fold.Growth of diploids in PFPA prior to exposure to the deaminating agent nitrous acid, the cross-linking agent mitomycin C, the alkylating chemical ethylmethanesulphonate (EMS) and UV light resulted in significant changes in the potency of these diverse mutagens to induce intragenic recombination. For all four mutagens, increased frequencies of gene convertants/viable cell were observed in those cultures which had been exposed to the amino acid analogue prior to mutagen treatment. In haploid WT yeast cells, amino acid analogue incorporation resulted in an enhanced frequency of UV induced forward mutation to canavanine resistance whilst in a DNA repair deficient rad 6 mutant this interaction between UV and PFPA was abolished.The results have been interpreted on the basis of incorporation of the analogue into enzymes involved with DNA replication with a consequent loss of fidelity of such enzymes and increased errors in base incorporation.     

The cross sensitivity to radiations, chemical mutagens and heat treatment of X-ray sensitive mutants of yeast

Summary A number of radiation sensitive mutants of yeast were examined for their sensitivity to the inactivating agents, ultraviolet light (UV), gamma irradiation, ethyl methane sulphonate (EMS) and heat treatment (52° and 37°).A mutant of the gene rad-3, isolated on the basis of its primary sensitivity to UV showed sensitivity only to UV. In contrast the five X-ray sensitive mutants were sensitive to all four inactivating treatments. Considerable variation was observed in the response of the mutants to liquid holding treatment in non-nutrient solution.The data concerning the heat sensitivity of the X-ray sensitive mutants confirms the correlation between heat and X-ray sensitivity observed in bacteria by Bridges (1969).The results indicate that at least two separable pathways of cellular repair exist in yeast, one effective in the repair of UV damage and the other effective in the repair of ionising radiation, alkylating agents, heat and a fraction of UV damage.     

Effect of elevated temperatures on the radiation sensitivity of yeast cells of different species

Summary The influence of hyperthermia on the survival of irradiated yeast cells of different species has been studied. The experiments reported in the paper have shown: (1) simultaneous action of ionizing radiation and high temperatures appeared to increase the radiation response by a factor of approximately 2.7 for diploid and only by a factor of 1.5 for haploid cells of wild-type; (2) the combined action of high temperature and ionizing radiation had no synergistic effect for rad51 mutant diploid yeast cells; (3) heating before or after irradiation did not alter the radiation response of yeast cells; (4) enhancement of yeast cell sensitivity by simultaneous action of hyperthermia and²³⁹Pu--particles was negligible; (5) the magnitude and the rate of liquid holding recovery is lowered with increasing of irradiation temperature. On this basis, it was concluded that possible mechanism for thermal sensitization of yeast cells may involve the reduced capacity of cells to recover damages resulted from the combined action of both modalities.     

RBE of densely ionizing radiation for wild-type and radiosensitive mutants of yeast

Survival curves were obtained for haploid and diploid yeasts, Saccharmyces cerevisiae, of wild-type strains and radiosensitive mutants exposed to γ-rays and α-particles. A correlation between the values of the relative biological effectiveness (RBE) of high-LET radiation and cell-repair capacity was found. The difference in radiosensitivities of the wild-type diploid strain and homozygous rad mutants incapable of recovery was significantly higher after low-LET radiation than after high-LET radiation. Possible reasons for the observed radiation responses to low- and high-LET exposure of yeast cells with various genotype are discussed.     

The oxygen effect in haploid and diploid yeast strains of different sensitivities

The extent of the oxygen effect for cell survival was studied in diploid and haploid wild-type yeast and in mutants beloning to the rad2, rad6 and rad50 epistatic group. In haploids, reduced oxygen enhancement ratios were found in rad52, rad6 and rad18. The latter two also showed some influence of the mating type. In diploids the oxygen effect was decreased in rad2, rad52 and rad18.     

Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts.

Liquid-holding conditions can be obtained for human diploid skin fibroblasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light or X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cell strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT5Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures.     

The oxygen effect on transcription inhibition in yeast

No oxygen effect is found if X-ray-induced inhibition of ribosomal RNA synthesis in yeast is assessed immediately after exposure. A 20-h liquid holding period, however, leads to a differential recovery in diploid cells which is markedly greater after irradiation in nitrogen. Haploid yeast exhibits a much smaller recovery and does not show any significant difference between exposures in air or nitrogen.     

Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae

The treatment of diploid cultures of yeast with ultraviolet light (UV), γ-rays, nitrous acid (NA) and ethyl methane sulphonate (EMS) results in increases in cell death, mitotic gene conversion and crossing-over. Acridine orange (AO) treatment, in contrast, was effective only in increasing the frequency of gene conversion. The individual mutagens were effective in the order UV > NA > γ-rays > AO > EMS. Prior treatment of yeast cultures in starvation medium produced a significant reduction in the yield of induced gene conversion.The results have been interpreted on the basis of a general model of mitotic gene conversion which involves the post-replication repair of induced lesions involving de novo DNA synthesis without genetic exchange. In contrast mitotic crossing-over appears to involve the action of a repair system independent from excision or post-replication repair which involves genetic exchange between homologous chromosomes.     

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains ofSaccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in theG ₁ stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.     

The modifying effects of strain and age on the mutagenic specificity of ultraviolet light in Neurospora crassa

Summary Two derivatives of K3/17 ad-3A 38701; inos 37401 of Neurospora crassa are described which show opposite specific reversional responses to UV. Both derivatives carry the same two auxotrophic alleles and appear to differ only in a single gene which influences the pattern of mutagen specificity. The differences between the derivatives only develop after the cultures have been aged for two to four weeks. Various possible explanations are considered.     

The timing of UV mutagenesis in yeast: continuing mutation in an excision-defective (rad1-1) strain.

Summary Mutation induction by ultraviolet light was studied in excision-defective (rad1-1) strains of Saccharomyces cerevisiae. Information about the timing of mutations in relation to postirradiation DNA replication was obtained. The experimental system involved pedigree analysis of G1 diploid cells and subsequent tetrad analysis of the mitotic segregants to detect mutations.The mutation pattern of rad1-1 strains differed from that of wild type in two respects: (1) few or none of the mutations affected both strands of the DNA, (2) mutations appeared as frequently in the second postirradiation mitotic generation as in the first.The data have led to the following conclusions about the mutation process in excision-defective (rad1-1) yeast: (a) Mutations are not fixed prior to the first postirradiation round of DNA replication. (b) Unexcised thymine dimers persist as mutagenic lesions through repeated postirradiation cell divisions. (c) Heteroduplex repair is involved in the mutation process. (d) Overlapping daughter-strand gaps are not a prerequisite for mutation. (e) The results provide no evidence that error-prone repair in this strain is inducible rather than constitutive. The data also show that (f) all 2-strand mutations (whole-colony mutants) induced in yeast by exposure to low levels of ultraviolet light are associated with excision repair, and that (g) essentially all lesions induced in excision-proficient strains have been excised at the time of the second round of postirradiation DNA synthesis.On leave of absence from the Department of Genetics, University of Edinburgh, West Mains Road, Edinburgh, Scotland EH9 3JN     

Liquid-holding and recovery from UV-induced damage in Eudorina elegans

Summary Eudorina elegans does not respond to liquid-holding or to postirradiation medium effects by changes in recovery.A decrease in survival ability is observed if a culture is starved prior to irradiation, or is incubated at 22°C rather than 32°C following UV irradiation. Eudorina loses the ability to photoreactivate UV damage within 10 to 48 h following irradiation, depending upon the pre-and post-UV culture conditions.The results are interpreted as indicating a failure of Eudorina elegans to carry out specific dark repair of UV damage. Some reactivation may occur during cellular DNA synthesis.Abbreviations used PR photoreactivation - LHR liquid holding recovery - LHP liquid holding protection - ERR excision-resynthesis-repair - BC complete medium - BM minimal medium - cfa colony forming ability - cfu colony forming units Supported by grants from the National Research Council of Canada # A4431.     

The effect of radiation sensitivity and cell stage on liquid holding response in Schizosaccharomyces pombe

Summary The response of the wild type strain and 20 different radiation sensitive mutants of S. pombe to liquid holding after ultraviolet irradiation was ivestigated. Three of the sensitive mutants tested showed appreciable liquid holding recovery, as opposed to the negative liquid holding effect observed in the wild type cells. One of these mutants is reported to be recombination-deficient while the other two have a normal recombination capability. Further experiments were carried out by using G₁ cells and ascospores to test the possible role of a recombinational type of repair pathway in the failure of wild type S. pombe to show liquid holding recovery. Data from such studies indicated that the negative liquid holding effect observed in the wild type cannot be ascribed to this particular pathway. This conclusion is further supported by the observation that caffeine which is believed to inhibit mainly the recombinational repair in this yeast, did not alter the negative liquid holding effect in the wild type. This observation implies that the caffeine-sensitive repair process occurs only in a rich medium and not in the non-nutrient solution. Data have been discussed as these relate to possible cause(s) of negative liquid holding effect in this organism.     

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.