The repair of DNA methylation damage in Saccharomyces cerevisiae

 The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways. Received: 13 June/24 July 1996     

Functional mitochondria are essential for Saccharomyces cerevisiae cellular resistance to bleomycin

 The antitumor activity of bleomycin is associated with its ability to produce DNA lesions. The cellular process that repairs bleomycin-induced DNA lesions is not entirely clear. To understand how these DNA lesions are repaired in eukaryotic cells, we used mini Tn3 : : LEU2 :: LacZ transposon mutagenesis to isolate yeast mutants that were hypersensitive to bleomycin. One of the mutants, HCY69, was characterized further and found to be 4- and 3-fold more sensitive, respectively, to bleomycin and hydrogen peroxide, as compared to the parent. The mutant displayed parental resistance to a variety of other DNA-damaging agents. Plasmid rescue and DNA sequence analysis revealed that the transposon interrupted the OXA1 gene, which encodes a protein required to process one of the subunits, cox II, of the cytochrome oxidase complex in mitochondria. A plasmid carrying the native OXA1 gene fully restored drug resistance to strain HCY69. Our data strongly suggest that functional mitochondria are required for cellular protection against the toxic effects of bleomycin. Received: 22 April/4 July 1996     

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2-Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Delta cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair.     

Newly synthesised DNA of high molecular weight in the yeast Saccharomyces cerevisiae

Summary Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster.     

Cellular and molecular effects of bleomycin are modulated by heat shock in Saccharomyces cerevisiae

To study some mechanisms underlying the stress responses in eukaryotic cells, we investigated the effect of heat shock (HS) on the induction of DNA double strand breaks as well as on potentially lethal and mutagenic events induced by the radiomimetic antibiotic bleomycin (BLM) in Saccharomyces cerevisiae. Haploid wild-type yeast cells in the logarithmic phase of growth were exposed to different concentrations of BLM (0–30 μg/ml, 1.5 h) without and with a previous HS (38°C, 1 h). Immediately after treatments, survival as well as mutation frequency were determined, and quantitative analysis of chromosomal DNA by laser densitometry were performed both immediately after treatments and after incubation of cells during different time intervals in liquid nutrient medium free of BLM. Our results indicate that HS induces resistance to potentially lethal and mutagenic effects of BLM. Quantitative analysis of chromosomal DNA performed immediately after treatments showed the same DNA fragmentation, either upon BLM as single agent or preceded by HS. However, HS pretreated cells incubated during 4 h in liquid nutrient medium free of BLM repaired DNA double strand breaks more efficiently as compared to non-pretreated cells. On this basis, we propose that the observed HS-induced resistance to BLM depends on a regulatory network acting after DNA-induced damage, which includes genes involved in DNA repair, HS response and DNA metabolism.     

UV-inducible proteins in Saccharomyces cerevisiae

Summary Two UV-inducible proteins have been detected in the yeast, Saccharomyces cerevisiae. The proteins have molecular weights of 78,000 Daltons and 23,000 Daltons. This induction is specific for UV-irradiation as exposure to X-rays, mitomycin C and heat shock does not result in the synthesis of the proteins. The larger (78 kD) protein is induced in various rad strains and in a ° cir° strain. Attempts are being made to isolate the genes coding for these inducible proteins.     

Chromosomal proteins in Saccharomyces cerevisiae

Summary Proteins were isolated from purified yeast chromatin and subjected to two-dimensional electrophoresis. The cellular and the chromosomal content of the major nonhistone proteins was measured. Two polypeptides of molecular weights 55,000 and 53,000, identified as and tubulin, and a polypeptide of molecular weight 63,000, associated with the nuclear DNA to a very high degree, account for nearly 50% of the nonhistone proteins present in chromatin. Only one tenth of the RNA polymerase subunit with the molecular weight of 23,000 was associated with nuclear DNA following chromatin purification in metrizamide gradients.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae

Summary Four types of mit ^– mutations induced with manganese are found in the following relative proportions: oxi3 ^– > cob-box ^– > oxi2 ^– oxi1 ^–1. The frequences of loss of their respective mit ⁺ alleles in manganese-induced rho ^–] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.     

Repair of ultraviolet light damage in Saccharomyces cerevisiae as studied with double- and single-stranded incoming DNAs

Summary Purified double- and single-stranded DNAs of the autonomously replicating vector M13RK9-T were irradiated with ultraviolet light (UV) in vitro and introduced into competent whole cells of Saccharomyces cerevisiae. Incoming double-stranded DNA was more sensitive to UV in excision repair-deficient rad2-1 cells than in proficient repair RAD ⁺ cells, while single-stranded DNA exhibited high sensitivity in both host cells. The results indicate that in yeast there is no effective rescue of UV-incoming single-stranded DNA by excision repair or other constitutive dark repair processes.     

Polypeptide chain termination in Saccharomyces cerevisiae

Summary The study of translational termination in yeast has been approached largely through the identification of a range of mutations which either increase or decrease the efficiency of stop-codon recognition. Subsequent cloning of the genes encoding these factors has identified a number of proteins important for maintaining the fidelity of termination, including at least three ribosomal proteins (S5, S13, S28). Other non-ribosomal proteins have been identified by mutations which produce gross termination-accuracy defects, namely the SUP35 and SUP45 gene products which have closely-related higher eukaryote homologues (GST1-h and SUP45-h respectively) and which can complement the corresponding defective yeast proteins, implying that the yeast ribosome may be a good model for the termination apparatus existing in higher translation systems.While the yeast mitochondrial release factor has been cloned (Pel et al. 1992), the corresponding cytosolic RF has not yet been identified. It seems likely, however, that the identification of the gene encoding eRF could be achieved using a multicopy antisuppressor screen such as that employed to clone the E. coli prfA gene (Weiss et al. 1984). Identification of the yeast eRF and an investigation of its interaction with other components of the yeast translational machinery will no doubt further the definition of the translational termination process.While a large number of mutations have been isolated in which the efficiency of termination-codon recognition is impaired, it seems probable that a proportion of mutations within this class will comprise those where the accuracy of A site codon-anticodon interaction is compromised: such defects would also have an effect on termination-codon suppression, allowing mis- or non-cognate tRNAs to bind stop-condons, causing nonsense suppression. The remainder of mutatoons affecting termination fidelity should represent mutations in genes coding for components of the termination apparatus, including the eRF: these mutations reduce the efficiency of termination, allowing nonsense suppression by low-efficiency natural suppressor tRNAs. Elucidation of the mechanism of termination in yeast will require discrimination between these two classes of mutations, thus allowing definition of termination-specific gene products.     

Time-dependent mitotic recombination in Saccharomyces cerevisiae

The time-dependent appearance of prototrophic recombinants between heterologously located artificial repeats has been studied in Saccharomyces cerevisiae. While initial prototrophic colony numbers from independent cultures were highly variable, additional recombinants were found to arise daily at roughly constant rates irrespective of culture. These late-appearing recombinants could be accounted for neither by detectable growth on the selective media nor by delayed appearance of recombinants present at the time of selective plating. Significantly, at no time did the distributions of recombinants fully match those expected according to the Luria-Delbruck model and, in fact, after the first day, the distributions much more closely approximated a Poisson distribution. Prototrophic recombinants accumulated not only on the relevant selective medium, but also on media unrelated to the acquired prototrophy.     

A rapid assay for mitochondrial DNA damage and respiratory chain inhibition in the yeast Saccharomyces cerevisiae.

There is a need for a rapid assay to identify agents that damage mitochondria because the mitochondrion may be an important target for numerous environmental mitotoxins. Certainly at least one chemotherapeutic regimen (CHOP therapy) that includes doxorubicin can induce cardiomyopathy through mitochondrial genotoxicity in cardiac muscle cells. Yeast cells (1.5 x 10(6)-10(7)) in water are spread on a YEPD plate, and, when the suspension of cells has dried, a small well (12 mm diameter) is cut into the agar; 200-400 microl of a solution of the presumptive mitochondrial genotoxin is placed in the well, and the plates are incubated for 2 days. The genotoxin forms a concentration gradient through the agar and affects the growing cells. An overlay containing tetrazolium chloride is added, and the plates are incubated for 6-24 hr. Respiring cells turn red, and nonrespiring cells, with damaged DNA or inhibited respiratory chains, that are adjacent to the well, are white. A white ring, or a more lightly colored red ring, around the well indicates the presence of cells with lowered respiratory activity which may be fully reversible when the mitochondrial genotoxin is removed. In preliminary experiments, doxorubicin (= adriamycin) shows strong activity with this assay; cyclophosphamide is negative, and 4-hydroxycyclophosphamide, a metabolite of cyclophosphamide, is weakly positive. Ethidium bromide, methotrexate, 5-fluorouracil, and 5-fluorocytosine also are mitochondrial genotoxins. Antifungal agents similar to 5-fluorocytosine and anthelmintic compounds such as pyrvinium iodide can be powerful mitochondrial genotoxins.     

Stationary-phase mitophagy in respiring Saccharomyces cerevisiae

The clearance of malfunctioning mitochondria is an important housekeeping function in respiring eukaryotic cells and plays a role in physiological homeostasis as well as in the progression of late-onset diseases. This clearance is thought to occur by a specific form of autophagic degradation called mitophagy. Although the mechanism of nonspecific macroautophagy is relatively well established, the selective autophagic degradation of mitochondria has only recently begun to receive significant attention. An important step toward elucidating the mechanism by which defective mitochondria are selected and degraded is the establishment of conditions under which mitophagy is induced. This review covers our current understanding of mitophagy in the model organism Saccharomyces cerevisiae and its modes of activation, with a focus on stationary-phase mitophagy-a form of mitophagy that holds promise as a potential quality control mechanism.