Highly tolerated amino acid substitutions increase the fidelity of Escherichia coli DNA polymerase I
Highly tolerated amino acid substitutions increase the fidelity of Escherichia coli DNA polymerase I
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DOI：
10.1074/jbc.m611294200
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发表时间：
2007-04-20
影响因子：
4.8
通讯作者：
Loeb, Lawrence A.
中科院分区：
文献类型：
--
作者：
Loh, Ern;Choe, Juno;Loeb, Lawrence A.
DNA聚合酶催化的DNA合成的保真度对维持基因组的完整性至关重要。已经观察到准确度提高的突变聚合酶(反变异体)，但这些突变聚合酶主要涉及核酸外切酶校对增加或聚合酶活性大幅下降。我们测定了DNA聚合酶对大肠杆菌DNA聚合酶I活性部位和不同片段中氨基酸替换的耐受性，并确定了这些替换对DNA合成保真度的影响。我们建立了一个DNA聚合酶I突变文库，在整个聚合酶结构域中进行了随机替换。这个随机的文库首先被选为活动。DNA聚合酶的重要性及其序列和结构的保守性表明很少的氨基酸替代是可以容忍的。然而，我们报告说，三分之二的单碱基替换是可以容忍的而不会失去活性，可塑性通常发生在进化上保守的区域。我们筛选了活性文库中的408个成员，以了解在表达突变聚合酶并携带第二个含有β-内酰胺酶报告基因的质粒的大肠杆菌中DNA合成保真度的变化。突变频率是野生型的1/1000到1000倍。产生抗突变表型的突变分布在整个聚合酶区域，其中12%聚集在M-螺旋中。我们证实，该片段中的单个突变会导致碱基辨别力增加。因此，这项工作确定M-螺旋是保真度的决定因素，并表明聚合酶可以容忍许多改变保真度的取代，而不会引起活性的重大变化。
Fidelity of DNA synthesis, catalyzed by DNA polymerases, is critical for the maintenance of the integrity of the genome. Mutant polymerases with elevated accuracy ( antimutators) have been observed, but these mainly involve increased exonuclease proofreading or large decreases in polymerase activity. We have determined the tolerance of DNA polymerase for amino acid substitutions in the active site and in different segments of E. coli DNA polymerase I and have determined the effects of these substitutions on the fidelity of DNA synthesis. We established a DNA polymerase I mutant library, with random substitutions throughout the polymerase domain. This random library was first selected for activity. The essentiality of DNA polymerases and their sequence and structural conservation suggests that few amino acid substitutions would be tolerated. However, we report that two-thirds of single base substitutions were tolerated without loss of activity, and plasticity often occurs at evolutionarily conserved regions. We screened 408 members of the active library for alterations in fidelity of DNA synthesis in Escherichia coli expressing the mutant polymerases and carrying a second plasmid containing a beta-lactamase reporter. Mutation frequencies varied from 1/1000- to 1000-fold greater compared with wild type. Mutations that produced an antimutator phenotype were distributed throughout the polymerase domain, with 12% clustered in the M-helix. We confirmed that a single mutation in this segment results in increased base discrimination. Thus, this work identifies the M-helix as a determinant of fidelity and suggests that polymerases can tolerate many substitutions that alter fidelity without incurring major changes in activity.