ERROR-PRONE REPLICATION OF REPEATED DNA-SEQUENCES BY T7 DNA-POLYMERASE IN THE ABSENCE OF ITS PROCESSIVITY SUBUNIT
ERROR-PRONE REPLICATION OF REPEATED DNA-SEQUENCES BY T7 DNA-POLYMERASE IN THE ABSENCE OF ITS PROCESSIVITY SUBUNIT
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DOI：
10.1073/pnas.91.15.6830
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发表时间：
1994-07-19
影响因子：
11.1
通讯作者：
JOHNSON, KA
中科院分区：
文献类型：
--
作者：
KUNKEL, TA;PATEL, SS;JOHNSON, KA
我们研究了硫氧还蛋白（一种赋予噬菌体 T7 DNA 聚合酶高持续合成能力的辅助蛋白）对 DNA 合成保真度的影响。在硫氧还蛋白存在的情况下，外切核酸酶丰富的 T7 DNA 聚合酶具有很高的准确性。在对恢复 M13mp2 lacZ α 互补突变体的错误进行评分的保真度测定中，碱基替换的错误率小于或等于 2.2 x 10(-6)，恢复 +1 和 -1 阅读框中突变的移码分别小于或等于 3.7 x 10(-7) 和小于或等于 4.5 x 10(-7)。缺乏 3' --> 5' 核酸外切酶活性的聚合酶.硫氧还蛋白复合物的合成过程中，速率高出 10 倍以上，这表明移码和取代错误需要校对。通过比较存在或不存在辅助蛋白的情况下核酸外切酶缺陷型聚合酶的保真度，检查了硫氧还蛋白对准确性的贡献。硫氧还蛋白可以增强或降低保真度，具体取决于所考虑的错误类型。在不存在硫氧还蛋白的情况下，T7 DNA 聚合酶对于碱基替换的准确度提高了 3 倍，对于非重复核苷酸序列中 1-nt 和 2-nt 缺失错误的准确度分别提高了 27 倍和 9 倍。所有三个错误的较高保真度可能反映出聚合酶在缺乏辅助蛋白的情况下无法继续从突变前中间体合成。与此形成鲜明对比的是，当聚合反应中不存在硫氧还蛋白时，在重复的DNA序列中添加一个或多个核苷酸的移码错误率增加了46倍。错误率随着重复序列长度的增加而增加，这与链滑动导致模板引物错位的模型一致。因此，重复序列的复制扩展在不存在复制辅助蛋白的情况下发生。
We have examined the effect of thioredoxin, an accessory protein that confers high processivity to bacteriophage T7 DNA polymerase, on the fidelity of DNA synthesis. In the presence of thioredoxin, exonuclease-proficient T7 DNA polymerase is highly accurate. In fidelity assays that score errors that revert M13mp2 lacZ alpha-complementation mutants, error rates are less than or equal to 2.2 x 10(-6) for base substitution and less than or equal to 3.7 x 10(-7) and less than or equal to 4.5 x 10(-7) for frameshifts that revert mutations in the +1 and -1 reading frames, respectively. Rates are more than 10-fold higher during synthesis by polymerase.thioredoxin complex lacking 3' --> 5' exonuclease activity, demonstrating that frameshift as well as substitution errors are subject to proofreading. The contribution of thioredoxin to accuracy has been examined by comparing the fidelity of the exonuclease-deficient polymerase in the presence or absence of the accessory protein. Thioredoxin either enhances or reduces fidelity, depending on the type of error considered. In the absence of thioredoxin, T7 DNA polymerase is 3-fold more accurate for base substitutions and greater than or equal to 27-fold and 9-fold more accurate, respectively, for 1- and 2-nt deletion errors at nonreiterated nucleotide sequences. Higher fidelity for all three errors may reflect the inability of the polymerase to continue synthesis from the premutational intermediates in the absence of the accessory protein. In marked contrast, the rate for frameshift errors wherein one or more nucleotides has been added to a repeated DNA sequence increases 46-fold when thioredoxin is absent from the polymerization reaction. The error rate increases as the length of the repeated sequence increases, consistent with a model where strand slippage creates misaligned template-primers. Thus, replicative expansion of repetitive sequences occurs in the absence of a replication accessory protein.