Muller E G
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Arch Biochem Biophys. 1995 Apr 20;318(2):356-61. doi: 10.1006/abbi.1995.1240.
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 6 x 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.
在缺乏两个硫氧还蛋白基因TRX1和TRX2的酿酒酵母突变体中，DNA复制受到损害。只有当活性位点包含氧化还原活性半胱氨酸时，Trx1p才能支持正常的DNA复制速率。两种Trx1p突变形式，一种含有Cys30Ser突变，另一种含有Cys30Ser突变并伴有Cys33Ser突变，均无法维持正常的DNA合成速率。硫氧还蛋白活性位点突变体完成一轮复制需要66分钟，而等基因野生型培养物则为18分钟。使用针对纯化的6xHis标签Trx1p产生的抗体进行的蛋白质印迹分析表明，两种Trx1p突变形式的含量与野生型蛋白相同。因此，突变蛋白无法促进DNA合成不是由于降解或表达不佳，而是由于其还原能力丧失。结果表明，最佳的DNA合成速率需要硫氧还蛋白的氧化还原功能。由于在硫氧还蛋白突变体中测得的脱氧核苷酸水平正常，因此硫氧还蛋白要么与核糖核苷酸还原酶一起参与将一小部分脱氧核苷酸引导至复制位点，要么硫氧还蛋白还原并激活复制机制中一个未鉴定的成分。
