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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.
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Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.

机译：使用高度易错的DNA聚合酶I在大肠杆菌中靶向基因进化。

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摘要

We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. Mutagenesis is maximal in cultures maintained at stationary phase. Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions. Mutagenesis extends at least 3 kb beyond the 400-500 nt reportedly synthesized by Pol I. We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 beta-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam. Three different mutations contribute to aztreonam resistance. Two are found in the extended-spectrum beta-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described. Our system of targeted mutagenesis in E. coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology. Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases.
机译：我们提出了一种针对目标基因进化在大肠杆菌中随机诱变的系统。为了提高DNA聚合酶I（Pol I）复制的错误率，我们在控制Pol I保真度的三个结构域中引入了点突变。体内易于出错的Pol I的表达可导致诱变强烈编码在Pol I依赖性质粒中的靶序列（每bp 8.1 x 10-4突变，增加80,000倍），相对于染色体，质粒优先序列。在固定阶段的培养中诱变最大。突变均匀分布，并显示各种碱基对取代，主要是过渡。诱变延伸超过Pol I合成的400-500 nt的至少3 kb。我们证明，容易出错的Pol I可通过产生能够水解第三个酶的TEM-1β-内酰胺酶突变体来产生具有不同特性的酶。代内酰胺抗生素，氨曲南。三种不同的突变导致氨曲南耐药。在临床分离物中最常发现的广谱β-内酰胺酶中发现了两个，而第三个（G276R）以前没有被描述过。我们在大肠杆菌中的定向诱变系统应该对合成化学，基因治疗和分子生物学等领域中基于酶的应用产生影响。考虑到聚合酶之间的结构保守性，这项工作也应为改变其他聚合酶的保真度提供参考。

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