Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination.
Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination.
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DOI：
10.1038/s41598-018-31585-1
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发表时间：
2018-08-30
影响因子：
4.6
通讯作者：
Ichihashi N
中科院分区：
文献类型：
--
作者：
Sakatani Y;Yomo T;Ichihashi N
构建人工细胞的一个主要挑战是建立一个递归的基因组复制系统，并结合基因组本身的基因表达。递归DNA复制的最简单方案之一是结合重组的环状DNA的滚环复制。在这项研究中，我们试图开发一个复制系统的基础上，使用自我编码的phi29 DNA聚合酶和外部提供的Cre重组酶。我们首次发现DNA聚合被Cre重组酶显著抑制。为了克服这个问题，我们进行了体外进化，并获得了一个进化的环状DNA，可以有效地复制重组酶的存在。我们还证明了在进化DNA的复制过程中，环状DNA是通过Cre重组酶重组而复制的。这些结果表明，进化的环状DNA可以通过自我编码的聚合酶的基因表达来复制自己。这项研究提供了一个简单的递归DNA复制系统在人工细胞中使用的一步。
A major challenge in constructing artificial cells is the establishment of a recursive genome replication system coupled with gene expression from the genome itself. One of the simplest schemes of recursive DNA replication is the rolling-circle replication of a circular DNA coupled with recombination. In this study, we attempted to develop a replication system based on this scheme using self-encoded phi29 DNA polymerase and externally supplied Cre recombinase. We first identified that DNA polymerization is significantly inhibited by Cre recombinase. To overcome this problem, we performed in vitro evolution and obtained an evolved circular DNA that can replicate efficiently in the presence of the recombinase. We also showed evidence that during replication of the evolved DNA, the circular DNA was reproduced through recombination by Cre recombinase. These results demonstrate that the evolved circular DNA can reproduce itself through gene expression of a self-encoded polymerase. This study provides a step forward in developing a simple recursive DNA replication system for use in an artificial cell.
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