Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution
Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution
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DOI：
10.1038/nbt974
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发表时间：
2004-06-01
影响因子：
46.9
通讯作者：
Holliger, P
中科院分区：
文献类型：
--
作者：
Ghadessy, FJ;Ramsay, N;Holliger, P
DNA聚合酶以极高的特异性识别它们的底物(1，2)，限制了非天然核苷酸的使用及其实现的应用。我们描述了一种扩大聚合酶底物范围的策略。通过选择扭曲3‘错配的延伸，我们获得了Taq DNA聚合酶的突变体，它不仅混杂地延伸错配，而且获得了在保持高催化转换率、加工性和保真度的同时处理不同范围的非规范底物的通用能力。与野生型酶不同的是，它们绕过了阻断损伤的区域，如基本位点、胸苷二聚体或碱基类似物5-硝基吲哚(3)，并进行了PCR扩增，将所有四个核苷酸三磷酸完全替换为硫代磷酸(4)，或将其中一个替换为同等的荧光染料标记的核苷酸三磷酸。正如我们所展示的具有高达20倍明亮荧光的微阵列探针所展示的那样，这种“简单”的聚合酶有立竿见影的效果。
DNA polymerases recognize their substrates with exceptionally high specificity(1,2), restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3' mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol(3) and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates(4) or the substitution of one with the equivalent fluorescent dye-labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.