FIDELITY OF DNA-POLYMERASE-I AND THE DNA POLYMERASE-I-DNA PRIMASE COMPLEX FROM SACCHAROMYCES-CEREVISIAE
FIDELITY OF DNA-POLYMERASE-I AND THE DNA POLYMERASE-I-DNA PRIMASE COMPLEX FROM SACCHAROMYCES-CEREVISIAE
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DOI：
10.1128/mcb.9.10.4447
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发表时间：
1989-10-01
影响因子：
5.3
通讯作者：
SUGINO, A
中科院分区：
文献类型：
--
作者：
KUNKEL, TA;HAMATAKE, RK;SUGINO, A
我们测定了酵母DNA聚合酶I （yPol I）合成DNA的保真度。为了确定聚合酶催化亚基以外的亚基是否会影响保真度，我们测量了用常规方法纯化的yPol I的准确性，这种方法产生的DNA聚合酶具有部分蛋白水解的催化亚基，没有相关的引物酶活性，而用免疫亲和层析纯化的yPol I的准确性，这种方法产生的聚合酶具有单个高分子量的催化亚基。以及三种额外的多肽和DNA引物酶活性。在对lacZ . α中聚合酶错误进行评分的分析中。M13mp2 DNA中的-互补基因，yPol I和yPol I-引物酶复合体产生单碱基替换，单碱基移位和更大的缺失。对于特定的错误和模板位置，两种形式的聚合酶在保真度上表现出可能高达10倍的差异。然而，总体误差频率和误差谱的结果表明，yPol I-DNA引物酶复合物不是高度准确的，而且，就像单独的聚合酶一样，它的保真度不足以解释体内低自发突变率。特异性数据还提出了通过直接重复机制解释非重复序列中的- 1碱基帧移位和某些复杂缺失的模型，其中包括异常环路合成。
We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ .alpha.-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain - 1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.