Radiation-sensitive Mutations Research Articles

Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae.

The SRS2 gene of Saccharomyces cerevisiae was identified through mutational analysis as a suppressor of radiation-sensitive mutations in the error-prone repair pathway and by a hyper-recombination phenotype. Comparison of the derived amino acid sequence revealed the gene to have high homology to the bacterial DNA helicases UvrD and Rep (Aboussekhra, A., Chanet, R., Zgaga, Z., Cassier-Chauvat, C., Heude, M., and Fabre, F. (1989) Nucleic Acids Res. 17, 7211-7219). We have purified the SRS2 protein from Escherichia coli extracts by tagging the SRS2 gene with 6 carboxyl-terminal histidine residues and overexpressing the tagged protein in a pET-3c vector. Extracts were passed over a metal-chelating affinity chromatography column followed by gel filtration to obtain an enriched protein fraction. Sephacryl gel filtration of pooled fractions containing the SRS2 protein yielded purified SRS2 protein by Coomassie Blue stain of SDS-polyacrylamide gel electrophoresis gels. The purified SRS2 protein was found to have in vitro DNA-dependent ATPase and DNA helicase activities. The polarity of the helicase activity was determined to be 3' to 5', the same polarity as that found for the UvrD and Rep proteins. The carboxyl-terminal region of the protein is shown to contain a sequence for nuclear localization. Expression of the SRS2 in yeast was examined and found to be extremely low.

Spontaneous mitotic recombination in yeast: the hyper-recombinational rem1 mutations are alleles of the RAD3 gene.

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.

Hyper-mutation caused by the reml mutation in yeast is not dependent on error-prone or excision repair

The reml mutations of Saccharomyces cerevisiae confer a semi-dominant hyper-recombination/hypermutation phenotype. Neither reml mutant allele has any apparent meiotic affect. We have examined spontaneous mutation in reml-2 strains and demonstrate that the reml-2 mutation, like reml-1, confers an average 10-fold increase in reversion and forward mutation rates. Unlike certain yeast rad mutations with phenotypes similar to reml, strains containing reml are resistant to MMS and only slightly UV sensitive at very high doses. To understand the mutator phenotype of reml, we have used a double-mutant approach, combining the reml mutation with radiation-sensitive mutations affecting DNA repair. Double mutants of reml-2 and a mutation in the yeast error-prone repair group ( rad6-1) or a mutation in excision repair ( radl-2 or rad4) maintain the hyper-mutation phenotype. Since mutation rates remain elevated in these double-mutant strains, it appears as if the mutations which occur in the presence of reml resemble spontaneous mutation since they do not require the action of a repair system.

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.

Temperature-sensitive DNA repair mutations in the cellular slime mould Dictyostelium discoideum

The nonallelic radB13 and radG5 DNA repair mutations lower the plating efficiency at 26.5 °C of unirradiated D. discoideum cells. Revertants selected on the basis of resistance to gamma rays or UV from strains bearing either the radB13 or radG5 mutations are no longer temperature-sensitive at 26.5 °C. Unlike the wild-type and radiation-resistant revertants, survival following UV irradiation is lower at 24.5 °C than at 17.0 °C or 21.0 °C in strains carrying either the radB13 or radG5 mutation. We conclude that the gene products of the radB and radG loci probably affect normal cell growth by affecting DNA metabolism. Seven radiation-sensitive mutations that effect six loci other than radB and radG do not have temperature-sensitive phenotypes.

Interactions between radiation-sensitive mutations in double-mutant haploids of Dictyostelium discoideum.

Haploid strains of Dictyostelium discoideum carrying radiation-sensitive mutations in both the radA and radC genes are more sensitive to UV light irradiation than the "additive" sensitivity of the single-mutant haploids. This synergistic interaction indicates that the radA and radC gene products are involved in two different pathways of repair following UV-induced DNA damage. Double-mutant haploids bearing mutations in both the radA and radB genes have the same sensitivity as the radB single mutant indicating that the radA and radB gene products are involved in the same repair pathway.

Genetic analysis of radiation-sensitive mutations in the slime mould Dictyostelium discoideum.

The linkage of two mutations leading to increased sensitivity to ultraviolet light and 60Co gamma rays was determined in the slime mould Dictyostelium discoideum using a genetic analysis based on the parasexual cycle. Diploids were selected from a mixture of radiation-sensitive, temperature-resistant and radiation-resistant, temperature-sensitive haploids on the basis of simultaneous radiation and temperature resistance. Analysis of drug-resistant haploid segregants of the heterozygous diploids indicated that one of the radiation-sensitive mutations, radA20, was linked to linkage group I whereas the other, radB13, was linked to the recently defined linkage group VI.

Repair systems in Saccharomyces

Summary Radiation-sensitive mutations of yeast in multiple-mutant combinations interact with one another to affect survival. From these interactions, and from the effects of these mutations on other genetic events such as recombination or mutation, it is possible to deduce a scheme representing the pathways by which repair is effected. The biochemical roles of these pathways can be determined by simple assay systems. For example, one pathway controlled by 4 loci is concerned with the early stages of excision of pyrimidine dialers. Some of the loci used in this investigation affect the rate and duality of UV-induced mutation and so indicate what part repair might play in this process.

Genetic characterization of recombination-deficient mutants of Bacillus subtilis.

Recombination-deficient (rec), radiation-sensitive mutations in Bacillus subtilis are grouped in at least seven distinct loci. Map positions are determined for six of these loci.