Directed evolution of polymerase function by compartmentalized self-replication
Directed evolution of polymerase function by compartmentalized self-replication
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DOI：
10.1073/pnas.071052198
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发表时间：
2001-04-10
影响因子：
11.1
通讯作者：
Holliger, P
中科院分区：
文献类型：
--
作者：
Ghadessy, FJ;Ong, JL;Holliger, P
我们描述了区室化自我复制（CSR），这是一种酶（尤其是聚合酶）定向进化的策略。 CSR 基于一个简单的反馈环路，该环路由仅复制其自身编码基因的聚合酶组成。区室化用于将个体自我复制反应彼此隔离。在这样的系统中，适应性增益直接（并成比例地）转化为编码基因的遗传放大。 CSR 在聚合酶的进化中具有新颖且有用的特性。通过使用三个 CSR 循环，我们获得了标签 DNA 聚合酶的变体，其热稳定性比野生型酶高 11 倍，或者对强效抑制剂肝素的耐受性增加了 130 倍以上。在聚合酶反应之前在 CSR 循环中插入一个额外的阶段，使其能够应用于聚合酶以外的酶。我们证明核苷二磷酸激酶和标签聚合酶可以形成这样一个基于相互催化的协作CSR循环，从而核苷二磷酸激酶产生其自身基因复制所需的底物。我们还发现，在 CSR 中，聚合酶基因本身朝着更有效的复制方向进化。因此，聚合酶基因及其编码的多肽协作以使选择后拷贝数最大化。 CSR 应该被证明对于酶的定向进化，特别是 DNA 或 RNA 聚合酶，以及模拟生命前进化和病毒复制的体外自我复制系统的设计和研究有用。
We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based an a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Tag DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Tag polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.