A Saccharomyces cerevisiae mutant defines a new locus essential for resistance to the antitumour drug bleomycin

The antitumor drug bleomycin can produce a variety of lesions in the cellular DNA by a free radical dependent mechanism. To understand how these DNA lesions are repaired, bleomycin-hypersensitive mutants were isolated from the yeast Saccharomyces cerevisiae. We report here the analysis of one mutant, DRY25, that showed extreme sensitivity to bleomycin. This mutant also exhibited hypersensitivity to hydrogen peroxide and t-butyl hydroperoxide, but showed no sensitivity to other DNA-damaging agents, including γ-rays, ultraviolet light, and methyl methanesulfonate. Subsequent analysis revealed that strain DRY25 was severely deficient in the repair of bleomycin-induced DNA lesions. Under normal growth conditions, DRY25 displayed a 3-fold increase in the frequency of chromosomal translocation that was further stimulated by 5- to 15-fold when the cells were treated with either bleomycin or hydrogen peroxide, but not by methyl methanesulfonate, as compared with the wild type. Genetic analysis indicated that the mutant defect was independent of the nucleotide excision, postreplication, or recombinational DNA-repair pathways. These data suggest that one conceivable defect of DRY25 is that it lacks a protein that protects the cell against oxidative damage to DNA. A clone that fully complemented DRY25 defect was isolated and the possible roles of the complementing gene are discussed.Key words: yeast, bleomycin, DNA repair, mutations.

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A Saccharomyces cerevisiae phleomycin-sensitive mutant, phl40, is defective in the RAD6 DNA repair gene

Canadian Journal of Microbiology ◽

10.1139/m96-164 ◽

1996 ◽

Vol 42 (12) ◽

pp. 1263-1266 ◽

Cited By ~ 6

Author(s):

Chuan Hua He ◽

Jean-Yves Masson ◽

Dindial Ramotar

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Anticancer Agent ◽

Radiation Treatment ◽

Dna Lesions ◽

Exact Nature ◽

Repair Gene ◽

Yeast Saccharomyces Cerevisiae ◽

Dna Repair Gene ◽

Cellular Dna

The antibiotic bleomycin is used as an anticancer agent for treating a variety of tumours. The antitumour effect of bleomycin is related to its ability to produce lesions such as apurinic/apyrimidinic sites and single- and double-strand breaks in the cellular DNA. Phleomycin is a structurally related form of bleomycin, but it is not used as an anticancer agent. While phleomycin can also damage DNA, neither the exact nature of these DNA lesions nor the cellular process that repairs phleomycin-induced DNA lesions is known. As a first step to understand how eukaryotic cells provide resistance to phleomycin, we used the yeast Saccharomyces cerevisiae as a model system. Several phleomycin-sensitive mutants were generated following γ-radiation treatment and among these mutants, phl40 was found to be the most sensitive to phleomycin. Molecular analysis revealed that the mutant phl40 harbored a mutation in the DNA repair gene RAD6. Moreover, a functional copy of the RAD6 gene restored full phleomycin resistance to strain phl40. Our findings indicate that the RAD6 protein is essential for yeast cellular resistance to phleomycin.Key words: yeast, phleomycin, DNA repair, RAD6.

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Overlapping Roles of the Cytoplasmic and Mitochondrial Redox Regulatory Systems in the Yeast Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.4.2.392-400.2005 ◽

2005 ◽

Vol 4 (2) ◽

pp. 392-400 ◽

Cited By ~ 60

Author(s):

Eleanor W. Trotter ◽

Chris M. Grant

Keyword(s):

Oxidative Stress ◽

Saccharomyces Cerevisiae ◽

Redox State ◽

Redox Homeostasis ◽

Thioredoxin Reductase ◽

Normal Growth ◽

Growth Conditions ◽

Reduced Form ◽

Yeast Saccharomyces Cerevisiae ◽

Thioredoxin System

ABSTRACT Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.

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The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway.

Genetics ◽

10.1093/genetics/124.4.817 ◽

1990 ◽

Vol 124 (4) ◽

pp. 817-831 ◽

Cited By ~ 6

Author(s):

R H Schiestl ◽

S Prakash ◽

L Prakash

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Gamma Ray ◽

Dna Lesions ◽

Recombinational Repair ◽

Repair Pathway ◽

Uv Mutagenesis ◽

Uv Sensitivity ◽

Suppressor Mutations ◽

Induced Mutagenesis

Abstract rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

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Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽

10.1093/genetics/123.1.81 ◽

1989 ◽

Vol 123 (1) ◽

pp. 81-95 ◽

Cited By ~ 1

Author(s):

E J Louis ◽

J E Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Chromosome ◽

Stop Codon ◽

Fold Increase ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Nonsense Mutations ◽

Chromosome Iii ◽

Meiosis I ◽

Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

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The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3827-3836.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3827-3836

Author(s):

N P Williams ◽

P P Mueller ◽

A G Hinnebusch

Keyword(s):

Translational Control ◽

Normal Growth ◽

Regulatory Elements ◽

Growth Conditions ◽

Open Reading Frames ◽

Regulatory Function ◽

Yeast Saccharomyces Cerevisiae ◽

Reading Frame ◽

Yeast Genes ◽

Reading Frames

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

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The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.

Acta Biochimica Polonica ◽

10.18388/abp.1999_4162 ◽

1999 ◽

Vol 46 (2) ◽

pp. 289-298 ◽

Cited By ~ 1

Author(s):

A Hałas ◽

Z Policińska ◽

H Baranowska ◽

W J Jachymczyk

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Uv Irradiation ◽

Dna Polymerases ◽

Relative Molecular Mass ◽

Yeast Saccharomyces Cerevisiae ◽

Strand Breaks ◽

Single Strand Breaks ◽

Mutant Strains ◽

Cellular Dna

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.

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Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3847-3853.1986 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3847-3853

Author(s):

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mechanisms ◽

Normal Growth ◽

Regulatory Elements ◽

Growth Conditions ◽

Physical Structure ◽

Base Pairs ◽

Inducible Promoters ◽

Upstream Promoter ◽

Selection Of

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.

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Identification and Characterization ofMAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

Journal of Bacteriology ◽

10.1128/jb.180.11.2875-2882.1998 ◽

1998 ◽

Vol 180 (11) ◽

pp. 2875-2882 ◽

Cited By ~ 101

Author(s):

Eckhard Boles ◽

Patricia de Jong-Gubbels ◽

Jack T. Pronk

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activity ◽

Pyruvate Kinase ◽

Malic Enzyme ◽

Fold Increase ◽

Growth Conditions ◽

Anaerobic Growth ◽

Oxidative Decarboxylation ◽

Mrna Levels ◽

Malic Enzyme Activity

ABSTRACT Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

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PURE AND MOSAIC CLONES — A REFLECTION OF DIFFERENCES IN MECHANISMS OF MUTAGENESIS BY DIFFERENT AGENTS IN SACCHAROMYCES CEREVISIAE

Canadian Journal of Genetics and Cytology ◽

10.1139/g81-009 ◽

1981 ◽

Vol 23 (1) ◽

pp. 73-79 ◽

Cited By ~ 11

Author(s):

A. Nasim ◽

M. A. Hannan ◽

Earle R. Nestmann

Keyword(s):

Saccharomyces Cerevisiae ◽

Ultraviolet Light ◽

Nitrous Acid ◽

Repair Process ◽

Dna Lesions ◽

Ethyl Methanesulfonate ◽

Methyl Methanesulfonate ◽

Mechanisms Of Mutagenesis ◽

G1 Cells

The induction of pure and mosaic clones has been studied in haploid G1 cells of Saccharomyces cerevisiae. Following treatments with ultraviolet light, methyl methanesulfonate, ethyl methanesulfonate, nitrous acid, and N-methyl-N′-nitro-N-nitrosoguanidine, the relative proportions of pure mutant clones varied from 25 to 100% at comparable survival levels. Ultraviolet light and methyl methanesulfonate produced mainly pure mutant clones, whereas ethyl methanesulfonate and nitrous acid produced mainly mosaics at 59 to 100% survival levels. The ratio of pure to mosaic clones induced by nitrosoguanidine fell between these two classes. These results are consistent with a classification of mutagens on the basis of repair and replication-dependent mechanisms of mutagenesis in other organisms. Agents having actions similar to ultraviolet light may produce mainly pure clones through a pre-replicative process involving an error-prone DNA repair process. Others may produce mainly mosaic mutants due to the different nature of DNA lesions which may require a replication-dependent process for fixation of mutations. Preliminary data from combined treatments of mutagens belonging to two different classes (i.e. ultraviolet light and nitrous acid) suggest the possibility of an interaction between these agents, resulting in a higher proportion of pure clones, possibly due to an inducible process. Studies of induced frequencies of pure and mosaic clones may be useful in the characterization of mutagens with functional differences.

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