Department of Bioengineering and Robotics, Tohoku University, 6-6-01, Aramakiaza-aoba, Aoba-ku, Sendai, Miyagi, 980-8579, Japan.
Nucleic Acids Res. 2013 Aug;41(14):7176-83. doi: 10.1093/nar/gkt489. Epub 2013 Jun 3.
Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription-translation-replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription-translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds.
所有活细胞的复制都依赖于中心法则的多轮流转。特别是，DNA 复制蛋白的表达是循环进行中心法则过程的关键步骤。在这里，我们通过自主表达重组转录-翻译-复制系统（PURE 系统）中的染色体 DNA 复制机制，实现了整个顺序转录-翻译-复制过程。我们发现，低温对于使用 PURE 系统在单个试管中表达复杂蛋白 DNA 聚合酶 III 是必不可少的。将 13 个基因（编码起始因子、DNA 解旋酶、解旋酶加载器、RNA 引物酶和 DNA 聚合酶 III）添加到 PURE 系统中，通过偶联方式形成了 DNA 复制系统。一个人工遗传回路表明，复制产生的 DNA 能够为蛋白质提供遗传信息，表明体外中心法则可以连续进行两轮。
