Abstract:
The Saccharomyces cerevisiae gene that encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, was cloned and sequenced. The gene (RNR2) was further characterized by determining its copy number and by S1 mapping its transcriptional start site. The RNR2 protein was shown to be induced by the DNA-damaging agent methyl methanesulfonate. This induction was found to occur at the level of transcription and was shown to be independent of protein synthesis. An RNR2/LACZ fusion was constructed and used to identify other DNA-damaging agents that induced the RNR2 gene. The regulatory elements that control RNR2 induction were identified by a deletion analysis of the upstream region of RNR2. These elements, which consisted of two activation sites and a repression sequence, were contained on a fragment of approx 200 bp that was found to confer MMS inducibility on the heterologous CYC1 gene. A known transcriptional activator, the RAP1 protein was shown to bind to the downstream activation site; but RAP1 overproduction or depletion did not significantly affect RNR2 expression. These observations along with the result that RAP1 was found to be constitutively bound in vivo suggested that the RAP1 protein contributes to RNR2 expression, but it probably does not respond directly to an inducing signal. When the RNR2 repression sequence was inserted between the CYC1 activation sequences and TATA elements, it decreased the expression of the CYC1 gene. In addition, this repression was found to depend on SSN6, a protein required for the repression of a variety of other yeast genes. The RNR2 gene was also found to be induced by cell division cycle mutations that deplete cells of precursors used for DNA synthesis, which suggested that DNA damage and/or the inhibition of DNA replication could induce RNR2. In addition, methyl methanesulfonate induction was found to be reduced in a rad9 mutant, which is defective in cell cycle arrest, an event that normally accompanies DNA damage. This result further confirmed the view that the regulation of RNR2 expression is associated with processes of DNA repair. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD92-04061).
Keywords: Cell Division DNA/BIOSYNTHESIS DNA Damage DNA Repair DNA Replication *Gene Expression Regulation, Fungal Gene Products, vpr/METABOLISM Genes, vpr/GENETICS Genes, Fungal/*GENETICS Lac Operon/PHYSIOLOGY Mutation Ribonucleotide Reductases/*GENETICS Saccharomyces cerevisiae/*GENETICS TATA Box THESIS
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