Deoxyribonucleotides are maintained at normal levels in a yeast thioredoxin mutant defective in DNA synthesis.
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.
Muller
E G
EG
Department of Biochemistry, University of Washington, Seattle 98195.
D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000235:genetics; D003854:Deoxyribonucleotides / Q000378:metabolism*; D009154:Mutation; D012264:Ribonucleotide Reductases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics*
D004271:DNA, Fungal; D003854:Deoxyribonucleotides; D013879:Thioredoxins; D012264:Ribonucleotide Reductases