Economizing DNA-specific labelling by deoxythymidine-5′-monophosphate in Saccharomyces cerevisiae

Fäth, Wolfgang W.; Brendel, Martin; Laskowski, Wolfgang; Lehmann-Brauns, Elke

doi:10.1007/bf00268573

Cited by 33 publications

(6 citation statements)

References 12 publications

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“…The in vitro activity of thymidylate synthetase from S. cerevisiae requires N5,N'0-methylenetetrahydrofolate (17). Concurrent treatment with aminopterin and sulfanilamide inhibits thymidylate biosynthesis (I3, 15,[21][22][23][24][25], presumably by depleting endogenous tetrahydrofolate pools (Fig. 1).…”

Section: Fig 3 Fdump-induced Expression Of Cycloheximide Resistancementioning

confidence: 99%

“…Drugs that inhibit this enzyme or that limit the supply of reduced folates can abolish dTMP biosynthesis. In yeast, the combination of aminopterin plus sulfonamide is a potent inhibitor (13,15,(21)(22)(23)(24)(25). Cells thus treated require thymidylate, adenine, histidine, and methionine for growth (13,15 Previous work had shown that dTMP deprivation in yeast was not mutagenic for nuclear genes, but caused mitochondrial point mutations and cytoplasmic petites (14).…”

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confidence: 99%

“…In general, the dihydrofolate (FH2) analogue aminopterin inhibits dihydrofolate reductase (reaction 2) whereas sulfanilamide, an analogue of p-aminobenzoate, poisons de novo folate synthesis (reaction 3) (29). In yeast, treatment with aminopterin plus sulfanilamide blocks dTMP biosynthesis (13,15,(21)(22)(23)(24)(25), presumably by removing FH4 and so reducing the level of N 5,N'0-methylenetetrahydrofolate. dTMP (tup-) were sensitive to FdUMP whereas S288C, a wild-type strain (TUP+), was not.…”

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confidence: 99%

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Induction of mitotic recombination in yeast by starvation for thymine nucleotides.

Kunz¹,

Barclay²,

Game³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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The biosynthesis of thymine nucleotides in Saccharomyces cerevisiae can be inhibited either by genetic lesions in the structural gene for thymidylate synthetase (TMP1) or by drugs that prevent the methylation of dUMP to dTMP. This methylation can be blocked by folate antagonists. We find that 5-fluoro-dUMP (FdUMP) is also an effective inhibitor in vivo. Inhibition of dTMP biosythesis by these three different routes causes thymineless death. In addition to being cytotoxic, we find that FdUMP is highly recombinagenic in yeast but does not induce nuclear gene mutations. Provision of exogenous dTMP eliminates this induced mitotic recombination and cell killing. Similar results were obtained when a thymineless condition was provoked in cells by antifolate drugs or by dTMP deprivation in strains auxotrophic for this nucleotide. These findings show that, in contrast to the situation in prokaryotes, starvation for thymine nucleotides in yeast induces genetic recombination but is not mutagenic.

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Section: Fig 3 Fdump-induced Expression Of Cycloheximide Resistancementioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Induction of mitotic recombination in yeast by starvation for thymine nucleotides.

Kunz¹,

Barclay²,

Game³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…In vivo studies of DNA replication in yeast are limited by the general unsuitability of thymidine as a precursor (15) and, in dTMP uptake or auxotrophic mutants (10,37), by the very slow equilibration of large nucleotide pools (19,24). This problem is avoided with in vitro systems by using deoxynucleoside triphosphate (dNTP) substrates for DNA synthesis.…”

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confidence: 99%

Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether

Oertel

Goulian

1979

J Bacteriol

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Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With p+ strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-lBp+) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Densitylabeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In p0 strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperaturesensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment.

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“…Consequently, yeast DNA has been labeled with other radioactive bases or nucleotides with the attendant problem of incorporation of label into ribonucleic acid. To overcome this problem, mutants have been isolated (1,2,5,6,15,20) after selection for growth when the de novo pathway for deoxythymidine 5'-monophosphate synthesis was blocked through the combined action of aminopterin, an inhibitor ofdihydrofolate reductase, and sulfanilamide, an inhibitor of folate synthesis (1,15,20). The use of these mutants has some drawbacks.…”

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Bromodeoxyuridine 5'-monophosphate incorporation into yeast nuclear and mitochondrial deoxyribonucleic acid

Leff¹,

Lam²

1976

J Bacteriol

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Standard laboratory yeast strains can be enriched for thymidine 5'-monophosphate (TMP) uptake derivatives that generate only a low percentage of respiratory-deficient colonies (petites) under inhibition of TMP biosynthesis. Such mutants incorporated bromodeoxyuridine 5'-monophosphate (BrdUMP) into both nuclear and mitochondrial deoxyribonucleic acid (mtDNA); however, they showed a selectivity for TMP over BrdUMP incorporation. The preferential incorporation of [3H]TMP or BrdUMP into mtDNA was strain dependent. The density increments after growth in the presence of BrdUMP reached 50 mg/ml for nuclear DNA and 22 mg/ml for mtDNA in CsCl gradients. Density shifts correspondisng to 4% bromouracil substitution were easily detected. Preliminary density transfer experiments confirm that mtDNA does not replicate in synchrony with nuclear DNA.

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Economizing DNA-specific labelling by deoxythymidine-5′-monophosphate in Saccharomyces cerevisiae

Cited by 33 publications

References 12 publications

Induction of mitotic recombination in yeast by starvation for thymine nucleotides.

Induction of mitotic recombination in yeast by starvation for thymine nucleotides.

Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether

Bromodeoxyuridine 5'-monophosphate incorporation into yeast nuclear and mitochondrial deoxyribonucleic acid

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