A transcription and translation-coupled DNA replication system using rolling-circle replication
A transcription and translation-coupled DNA replication system using rolling-circle replication
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DOI：
10.1038/srep10404
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发表时间：
2015-05-27
影响因子：
4.6
通讯作者：
Yomo, Tetsuya
中科院分区：
文献类型：
--
作者：
Sakatani, Yoshihiro;Ichihashi, Norikazu;Yomo, Tetsuya
所有活生物体都具有基因组复制系统，其中基因组DNA通过从基因组转录的mRNA翻译的DNA聚合酶复制。这种基因组复制系统的人工重建是体外合成生物学的一大挑战。在这项研究中，我们试图构建一个转录和复制耦合的DNA复制（TTcDR）系统使用环状基因组DNA编码的phi29 DNA聚合酶和重建的转录和翻译系统。在该系统中，phi29 DNA聚合酶从基因组翻译并以滚环方式复制基因组。当使用传统的翻译系统组合物时，几乎没有观察到DNA复制，因为翻译系统中包含的tRNA和核苷三磷酸显著抑制DNA复制。为了最大限度地减少这些抑制作用，我们优化了TTcDR系统的组成，并将复制提高了约100倍。使用我们的系统，基因组DNA在30摄氏度下在12小时内复制了10次。该系统为体外构建人工基因组复制系统迈出了一步，这是构建人工细胞的先决条件。
All living organisms have a genome replication system in which genomic DNA is replicated by a DNA polymerase translated from mRNA transcribed from the genome. The artificial reconstitution of this genome replication system is a great challenge in in vitro synthetic biology. In this study, we attempted to construct a transcription-and translation-coupled DNA replication (TTcDR) system using circular genomic DNA encoding phi29 DNA polymerase and a reconstituted transcription and translation system. In this system, phi29 DNA polymerase was translated from the genome and replicated the genome in a rolling-circle manner. When using a traditional translation system composition, almost no DNA replication was observed, because the tRNA and nucleoside triphosphates included in the translation system significantly inhibited DNA replication. To minimize these inhibitory effects, we optimized the composition of the TTcDR system and improved replication by approximately 100-fold. Using our system, genomic DNA was replicated up to 10 times in 12 hours at 30 degrees C. This system provides a step toward the in vitro construction of an artificial genome replication system, which is a prerequisite for the construction of an artificial cell.