Caffeine enhancement of radiation killing in different strains of Saccharomyces cerevisiae.

Summary Haploid and diploid wild type strains, and three classes of radiation-sensitive mutants of Saccharomyces cerevisiae were tested for enhancement of UV-inactivation by caffeine in growth medium. In addition, the sensitizing effect of caffeine was studied in a haploid and a diploid wild type strain after gamma-irradiation. The drug sensitized the UV-irradiated cells of all strains except those reported to be only slightly UV-sensitive but highly sensitive to ionizing radiation. After gamma-irradiation, no caffeine-enhancement of killing was observed in stationary phase cells of either the haploid or the diploid strain. However, log-phase cells of both strains were partially sensitized.The results of both sets of experiments suggested that caffeine interferes with a recombinational repair occurring in cells in S or G2 phase.     

Liquid holding recovery kinetics in wild-type and radiosensitive mutants of the yeast Saccharomyces exposed to low- and high-LET radiations

Three wild-type diploid yeast strains Saccharomyces ellipsoideus and Saccharomyces cerevisiae and five radiosensitive mutants of S. cerevisiae in the diploid state were irradiated with gamma-rays from 60Co and alpha-particles from 239Pu in the stationary phase of growth. Survival curves and the kinetics of the liquid holding recovery were measured. It was shown that the irreversible component was enhanced for the densely ionizing radiation in comparison to the low-LET radiation while the probability of the recovery was identical for both the low- and high-LET radiations for all the strains investigated. It means that the recovery process itself is not damaged after densely ionizing radiation and the enhanced RBE of the high-LET radiation may be caused by the increased yield of the irreversible damage. A parent diploid strain and all its radiosensitive mutants showed the same probability for recovery from radiation damage. Thus, the mechanism of the enhanced radiosensitivity of the mutant cells might not be related to the damage of the repair systems themselves but with the production of some kind of radiation damage from which cells are incapable to recover.     

X-ray survival characteristics and genetic analysis for nine Saccharomyces deletion mutants that show altered radiation sensitivity

The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X rays, we are screening these mutants to identify additional genes that cause increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype cosegregates with the deletion allele and are obtaining multipoint survival-vs.-dose assays in at least one homozygous diploid and two haploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1, and VID21/EAF1 and discuss their potential roles in repair. Eight of these genes cause a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, results in at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultraviolet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino acids are also X-ray sensitive, which confirms that methylation of the lysine-79 residue is required for effective repair of radiation damage.     

The requirement for ATP hydrolysis by Saccharomyces cerevisiae Rad51 is bypassed by mating-type heterozygosity or RAD54 in high copy

Rad51 can promote extensive strand exchange in vitro in the absence of ATP hydrolysis, and the Rad51-K191R mutant protein, which can bind but poorly hydrolyze ATP, also promotes strand exchange. A haploid strain expressing the rad51-K191R allele showed an equivalent sensitivity at low doses of ionizing radiation to rad51-K191A or rad51 null mutants and was defective in spontaneous and double-strand break-induced mitotic recombination. However, the rad51-K191R/rad51-K191R diploid sporulated and the haploid spores showed high viability, indicating no apparent defect in meiotic recombination. The DNA repair defect caused by the rad51-K191R allele was suppressed in diploids and by mating-type heterozygosity in haploids. RAD54 expressed from a high-copy-number plasmid also suppressed the gamma-ray sensitivity of rad51-K191R haploids. The suppression by mating-type heterozygosity of the DNA repair defect conferred by the rad51-K191R allele could occur by elevated expression of factors that act to stabilize, or promote catalysis, by the partially functional Rad51-K191R protein.     

Studies on an unstable phenotype induced by UV irradiation

Unstable clones excreting L-lysine into their growth medium are obtained at a very high frequency following UV irradiation in both haploid and diploid strains of Saccharomycopsis lipolytica, provided they carry a mutation affecting the first enzyme of the lysine pathway and confering resistance to end product inhibition. The phenotype can be stabilized in some sublines; it appears as dominant and coupled with a decrease in spore viability. Excretion in batch cultures is confined to the end of the exponential phase, and seems not to consist in a simple release of the lysine pool content.     

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.     

Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae

Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cervisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MAT locus. The difference in levels of stimulation between MATa/MATα diploid and MATα haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m²), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MATα gene was introduced by DNA transformation into a MATa/matα::LEU2 ⁺ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATA gene was introduced by DNA transformation into a MATα haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.     

Mutants of Yeast Sensitive to Ultraviolet Light

Six uvr mutants of Saccharomyces cerevisiae with hypersensitivity to ultraviolet (UV) light were isolated after mutagen treatment with ethylmethanesulfonate. UV sensitivity ranges from moderate to extreme, and four of the mutants are also sensitive to nitrous acid. Ranking in terms of UV sensitivity does not parallel ranking in terms of nitrous acid sensitivity. Homozygous diploid mutant strains are somewhat less sensitive than the corresponding haploids. All mutations are recessive. None of the mutants is sensitive to gamma rays, and each shows photoreactivation after UV radiation. Complementation tests and tetrad analysis indicate that each strain represents mutation in a different gene. Two of the uvr genes are linked, and two others are centromere-linked.     

Dependence of the radiosensitivity of yeast cells on radiation LET. A theoretical analysis

A model is proposed for interpreting the radiosensitivity of yeast cells as a function of linear energy transfer (LET) of ionizing radiation. The model takes into account the role of repair processes in sensitivity of yeast cells to ionizing radiation of different LET. Two types of repair are discussed: (1) a nonspecific repair (characteristic of both haploid and diploid cells), and (2) a diploid-specific repair (characteristic of diploid cells only).     

Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme.

We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.     

Allelic and Ectopic Interactions in Recombination-Defective Yeast Strains

Ectopic recombination in the yeast Saccharomyces cerevisiae has been investigated by examining the effects of mutations known to alter allelic recombination frequencies. A haploid yeast strain disomic for chromosome III was constructed in which allelic recombination can be monitored using leu2 heteroalleles on chromosome III and ectopic recombination can be monitored using ura3 heteroalleles on chromosomes V and II. This strain contains the spo13-1 mutation which permits haploid strains to successfully complete meiosis and which rescues many recombination-defective mutants from the associated meiotic lethality. Mutations in the genes RAD50, SPO11 and HOP1 were introduced individually into this disomic strain using transformation procedures. Mitotic and meiotic comparisons of each mutant strain with the wild-type parental strain has shown that the mutation in question has comparable effects on ectopic and allelic recombination. Similar results have been obtained using diploid strains constructed by mating MATa and MAT alpha haploid derivatives of each of the disomic strains. These data demonstrate that ectopic and allelic recombination are affected by the same gene products and suggest that the two types of recombination are mechanistically similar. In addition, the comparison of disomic and diploid strains indicates that the presence of a chromosome pairing partner during meiosis does not affect the frequency of ectopic recombination events involving nonhomologous chromosomes.     

Comparative study of RBE of densely ionizing radiation for various types of cell death in yeast

A comparative study of the relative biological effectiveness (RBE) of alpha-particles 249Pu for reproductive and interphase forms of killing of haploid and diploid yeast cells of wild-type and their radiosensitive mutants has been carried out. The correlation between the RBE of alpha-particles and cell repair capacity was confirmed for reproductive death: it was the highest for diploid cells, smaller for haploid cells and the smallest for their radiosensitive mutants. To achieve the interphase cell killing much higher irradiation doses were used after which cells were incapable of liquid-holding recovery during the storing of exposed cells in non-nutrient media at 30 degrees C. The RBE values for this form of killing were significantly lower in comparison with reproductive death. These data are an additional argument supporting the point of view that the RBE of densely ionizing radiation is determined not merely by physical processes of energy absorption as it is traditionally believed but also by ability of cells to recover from DNA damages inflicted by ionizing radiation.     

DNA polymerase 4 of Saccharomyces cerevisiae is important for accurate repair of methyl-methanesulfonate-induced DNA damage

The DNA polymerase 4 protein (Pol4) of Saccharomyces cerevisiae is a member of the X family of DNA polymerases whose closest human relative appears to be DNA polymerase lambda. Results from previous genetic studies conflict over the role of Pol4 in vivo. Here we show that deletion of Pol4 in a diploid strain of the SK1 genetic background results in sensitivity to methyl methanesulfonate (MMS). However, deletion of Pol4 in other strain backgrounds and in haploid strains does not yield an observable phenotype. The MMS sensitivity of a Pol4-deficient strain can be rescued by deletion of YKu70. We also show that deletion of Pol4 results in a 6- to 14-fold increase in the MMS-induced mutation frequency and in a significant increase in AT-to-TA transversions. Our studies suggest that Pol4 is critical for accurate repair of DNA lesions induced by MMS.     

Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays

A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of α-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene–gene interactions.     

Aberrations in sexual behavior in some brewing and other industrial yeasts

Summary The mating behavior of a number of brewer's and distiller's yeasts was determined with a and haploid and aa and diploid tester strains. Mating frequencies were not high, ranging from one to (rarely) 2,000/10⁸ cells in the mating mixture. Sporulating hybrids were obtained in most matings, though the percentage spore viability initially obtained was often low. Notable the spore viability obtained in hybrids with the haploid tester strains and the brewing strains DIB and DICH was much higher than from the a haploid tester strain, and higher in hybrids between these strains and the aa diploid tester than in those from the tester strain. With the brewing strain NBA, the spore viability in hybrids with the a haploid tester strain was higher than in the case of strains DIB and DICH, but the spore viability in the hybrid of NBA x the haploid strain was higher still. The data are consistent with the hypothesis that with the a and aa tester strains, most of the industrial yeasts tested mate as diploids, and with the and testers, they mate as haploids, an hypothesis which is supported by the segregation of adenine markers in the progeny of these hybrids.Presented in part at the 6th International Specialized Symposium on Yeasts, Montpellier, France, 2–8 July, 1978     

NUCLEAR-CYTOPLASMIC RELATIONS IN THE MITOSIS OF SEA URCHIN EGGS : III. γ-Ray-Induced Damage to Whole Eggs and Nucleate and Anucleate Half-Eggs

Sea urchin eggs were cut into halves. The nucleate and anucleate halves and whole eggs were irradiated with γ-rays and then fertilized with normal sperm. The first mitosis of the diploid half-egg was more delayed than the division of the whole egg. There was a small, but highly significant, delay of the mitosis of the haploid half-egg, thus demonstrating cytoplasmic sensitivity to ionizing radiation. Since the sensitivity of nucleate cells is influenced by cytoplasmic volume, the problem of the role of cytoplasm in repair is considered in relation to these data and other reports in the literature.     

Mutation frequency of Dictyostelium discoideum spores exposed to the space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiosensitive mutant and the parental wild-type strain, were used to investigate the effects of cosmic radiation on viability and mutation frequency at the spore stage for about 9 days in Space Shuttle of NASA. We measured little effect of space environment on viability and cell growth in the both strains as compared to ground controls. The mutation frequency of the flown spores were similar to that of ground control. These results suggest that there could be no effect of cosmic radiation, containing high linear energy transfer radiation at about 0.9 mSv/day as detected by real-time radiation monitoring device on the induction of mutation at the spore stage.     

Mutability of the LYS2 gene in diploid saccharomycete yeasts. I. The occurrence of spontaneous mutants and mutants induced by x-ray and ultraviolet radiation

More than 3000 spontaneous and induced lys2 mutants were obtained in haploid and diploid strains of yeast Saccharomyces. The ability to utilize alpha-aminoadipate was used for lys2 mutant screening. The spontaneous and induced mutation rates were measured in haploid and diploid strains. Mitotic segregation of pho1 marker linked to LYS2 was studied in lys2 mutants obtained in diploid strains. Fertility of diploid lys2 mutants was tested. The conclusion to be drawn from the data presented is that mutations appeared in one of two homologous chromosomes and then segregated by mitotic homozygotization.     

Single-gene deletions that restore mating competence to diploid yeast

Using the Saccharomyces cerevisiae MATa/MATalpha ORF deletion collection, homozygous deletion strains were identified that undergo mating with MATa or MATalpha haploids. Seven homozygous deletions were identified that confer enhanced mating. Three of these, lacking CTF8, CTF18, and DCC1, mate at a low frequency with either MATa or MATalpha haploids. The products of these genes form a complex involved in sister chromatid cohesion. Each of these strains also exhibits increased chromosome loss rates, and mating likely occurs due to loss of one copy of chromosome III, which bears the MAT locus. Three other homozygous diploid deletion strains, ylr193cDelta/ylr193cDelta, yor305wDelta/yor305wDelta, and ypr170cDelta/ypr170cDelta, mate at very low frequencies with haploids of either or both mating types. However, an ist3Delta/ist3Delta strain mates only with MATa haploids. It is shown that IST3, previously linked to splicing, is required for efficient processing of the MATa1 message, particularly the first intron. As a result, the ist3Delta/ist3Delta strain expresses unbalanced ratios of Matalpha to Mata proteins and therefore mates with MATa haploids. Accordingly, mating in this diploid can be repressed by introduction of a MATa1 cDNA. In summary, this study underscores and elaborates upon predicted pathways by which mutations restore mating function to yeast diploids and identifies new mutants warranting further study.     

Mitotic Recombination in Yeast: Isolation and Characterization of Mutants with Enhanced Spontaneous Mitotic Gene Conversion Rates

Semi-dominant mutants displaying greatly elevated (up to 200-fold above control) levels of spontaneous mitotic recombination have been isolated in a disomic haploid strain of yeast heteroallelic at the arg4 locus. They are designated by the symbol MIC. The mutants variously exhibit associated sensitivity to UV and ionizing radiation and to methyl methanesulfonate, enhanced UV-induced mitotic recombination, and enhanced spontaneous forward mutation rates. Possible enzyme defects and involvement in repair and editing of DNA are discussed. The mutants are expected to simplify the analysis of recombination pathways in yeast.