title: A transcription and translation-coupled DNA replication system using rolling-circle replication.
abstract: 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 °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.
authors.affiliation: Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sakatani
authors.affiliation: 1] Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan [2] Exploratory Research for Advanced Technology, Japan Science and Technology Agency. 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Norikazu
authors.initials: N
authors.lastname: Ichihashi
authors.affiliation: Exploratory Research for Advanced Technology, Japan Science and Technology Agency. 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kazuta
authors.affiliation: 1] Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan [2] Exploratory Research for Advanced Technology, Japan Science and Technology Agency. 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan [3] Graduate School of Frontier Biosciences, Osaka University University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
chemical_list: D004270:DNA, Circular; D004259:DNA-Directed DNA Polymerase