A redox-dependent function of thioredoxin is necessary to sustain a rapid rate of DNA synthesis in yeast.
A redox-dependent function of thioredoxin is necessary to sustain a rapid rate of DNA synthesis in yeast.
复制标题
硫氧还蛋白的氧化还原依赖性功能对于维持酵母中 DNA 的快速合成是必要的。
DOI：
10.1006/abbi.1995.1240
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Muller,EG
中科院分区：
文献类型：
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作者：
Muller,EG
DNA复制在缺少两个硫氧还蛋白基因TRX1和TRX2的酿酒酵母突变体中受损。只有当活性位点含有氧化还原活性半胱氨酸时，Trx1p才支持正常的DNA复制速率。Trx1p的两种突变形式，一种含有Cys30Ser突变，另一种含有Cys30Ser突变和Cys33Ser突变，无法维持正常的DNA合成速率。硫氧还蛋白活性位点突变体在66分钟内完成了一轮复制，而同基因野生型培养物则观察到18分钟。使用针对纯化的6× His标记的Trx1p产生的抗体进行的Western印迹分析表明，Trx1p的两种突变形式与野生型蛋白质的水平相同。因此，突变蛋白不能促进DNA合成不是由降解或表达不良引起的，而是由其还原能力的丧失引起的。结果表明，DNA合成的最佳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糖核苷酸水平是正常的，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糖核苷酸引导到复制位点，要么硫氧还蛋白减少并从而激活复制机制的未鉴定组分。
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× 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.