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Among the best studied examples of DNA amplification, as a response to drug selection, is the increased DHFR gene copy number observed in tumor cells resistant to methotrexate (MTX)1,2,. In spite of numerous studies into the mechanism of DHFR gene amplification in a variety of cell types, we do not yet have a detailed understanding of the molecular events which initiate the amplification process. Moreover, in spite of a relative wealth of information about the distribution and arrangement of amplicons observed in MTXR cells, we know very little about endogenous factors which affect the frequency of DHFR amplification-nor do we understand, at the molecular level, the process of resolution of early events into the final amplicon structures observed in drug-resistant cells.

Unlike wild-type strains of S. cerevisiae which are resistant to growth inhibition by trimethoprim (TRM), two mutants defective in error-prone DNA repair are sensitive to the drug1,2,3. Since first reported almost twenty years ago, the sensitivity of rad6 and rad18 mutants to TRM has been puzzling, in the absence of any apparent connection between folate metabolism and gene products involved in DNA repair. A role for a reduced folate in light-dependent DNA repair was first suggested when 5,10-methenyltetrahydrofolate (MTHF) was found to be associated with purified photolyases4,5 from E.coli and S.cerevisiae. In vitro studies, designed to define the function of the folate chromophore in photoreactivation, have led to the suggestion that MTHF acts as a light-gathering “antenna” of the enzyme6. Subsequently, it was found that yeast cells depleted of reduced folates are defective in both light and dark DNA repair in vivo 7,8. This has suggested a more general involvement of tetrahydrofolates in the repair response of yeast cells to the DNA damage induced by UV light.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

High concentrations of deoxythymidine monophosphate (dTMP) induce mutations in Saccharomyces cerevisiae. Strains defective in the RAD6 gene-thought to be involved in error-prone DNA repair-do not show dTMP-induced mutation. We propose a model to explain these findings and suggest that fluxes of thymidine nucleotides may diminish the fidelity of DNA replication.

During early meiotic development the yeast Saccharomyces cerevisiae has a characteristic nuclear dense body (NDB). It is shown that the NDB can also be induced in vegetatively growing cells through the inhibition of thymidylate synthetase which causes depletion of the dTMP pool and arrests DNA synthesis. The observations on NDBs and recombination levels suggest that thymidylate-stressed cells may activate parts of the meiotic pathway and, conversely, cells on sporulation medium may sense, among other things, reduced thymidylate levels and respond to the several stimuli by entering the meiotic pathway.

A rapid procedure for the recovery of Saccharomyces cerevisiae auxotrophs was developed by exploiting the protection of these mutants from thymineless death when a required metabolite was withheld. The method can be used for thymidine 5'-monophosphate-requiring auxotrophs or wild-type strains blocked in de novo synthesis of thymidylate by folate antagonists.