Abstract
Deletion of both thioredoxin genes TRX1 and TRX2 of Saccharomyces cerevisiae reduces the rate of DNA replication. This observation, originally determined by flow cytometry, was confirmed by radiochemical labeling of synchronized cultures. Since thioredoxin is a hydrogen donor to ribonucleotide reductase, a priori the inhibition of DNA synthesis was predicted to be caused by a reduction in the deoxyribonucleotide pools. However, the levels of TTP, dCTP, dATP, and dGTP were either unchanged or slightly greater in the thioredoxin mutant (3.2, 0.91, 1.4, and 1.21 pmol/10(6) cells, respectively) versus the wild-type culture (2.5, 0.91, 1.0, and 0.68 pmol/10(6) cells, respectively). An impact on ribonucleotide reduction was seen by an increased accumulation of RNR1 and RNR2 transcripts in the thioredoxin mutant (4.3- and 6.8-fold, respectively). Increased RNR expression did not reflect a general response of the DNA replication machinery. POL1 (DNA polymerase I) and CDC8 (thymidylate kinase) transcription were unaltered, while histone H2B transcripts actually decreased by half. Two alternative models incorporating these results are discussed. One suggests that thioredoxin reduces a multiprotein complex channeling nucleotides to the replication apparatus. The second proposes that thioredoxin regulates the tempo of DNA replication directly by activating a component of the replication machinery.
Highlights
Thioredoxin was originally defined as a hydrogen donor for ribonucleotide reductase (reviewed by Reichard (1988)).Thioredoxin reduces a pair of cysteines at the surfaceof ribonucleotide reductase, which shuttles the electrons tthoe active site for reduction of ribonucleotides to theircorresponding deoxyribonucleotide (Aberget al., 1989; Mao et al, 1992)
This paper addresses the questionof whether the slow rate of DNA synthesis in the yeastht ioredoxin double mutant derives from a reduced pool of dNTPs
DNA Synthesis Is InhibitedintheThioredoxinMutantPreviously reported results, which combined growth rates and flow cytometry analysis, demonstrateda %fold reduction in the rate of DNA synthesis in exponential cultures of a yeast thioredoxin double mutant deleted for both the TRXl and TRX2 genes (Muller, 1991)
Summary
All yeast strains used in this study were derived from W303 (Wallis et al, 1989) and EMY54 (Muller, 1992). The HPLC analysis separated several other unknown compoundswith retention times similar to the dNTPs under study Identification of these compounds wasoutside of the scope of the present work. Recovery of these unknown compounds was unaffectedby longer boronate fractionation, more extensive periodate treatment, or different timing of the methylamine addition.All these steps are designed to remove ribonucleotides from the extract, making it unlikely that the unknowns are ribonucleotide derivatives. Eleven extracts of both EMY6O and EMY63 were examined over the course of establishing this protocol.
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