A REDOX-DEPENDENT FUNCTION OF THIOREDOXIN IS NECESSARY TO SUSTAIN A RAPID RATE OF DNA-SYNTHESIS IN YEAST

DNA replication is impaired in mutants of Saccharomyces cerevisiae which lack the two thioredoxin genes TRX1 and TRX2. Trx1p supports a normal rate of DNA replication only if the active site contains the redox active cysteines. Two mutant forms of Trx1p, one containing a Cys30Ser mutation and a second containing the Cys30Ser mutation in combination with a Cys33Ser mutation, were unable to sustain normal rates of DNA synthesis. The thioredoxin active-site mutants completed a round of replication in 66 min as opposed to 18 min observed for an isogenic wild type culture. Western blot analysis, using antibody generated against purified 6x His-tagged Trx1p, showed that both mutant forms of Trx1p were present at the same levels as the wild-type protein. Thus the inability of the mutant proteins to promote DNA synthesis is not caused by degradation or poor expression, but rather by the loss of their reductive capacity. The results show that an optimal rate of DNA synthesis requires a redox function of thioredoxin. Since the measured levels of deoxyribonucleotides are normal in the thioredoxin mutants, thioredoxin either participates with ribonucleotide reductase in channeling a small subset of deoxyribonucleotides to sites of replication, or thioredoxin reduces and thereby activates an unidentified component of the replication machinery. (C) 1995 Academic Press, Inc.