Chromosome replication machineries of Escherichia coli generated by a reconstituted protein expression system

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Kei Fujiwara, Tsutomu Katayama, Shin-ichiro M. Nomura

Tohoku university, Japan

Replication of all living cells relies on the multi-rounds flow of the central dogma, and reconstituting this flow is an important step in both understanding and in creating self-reproducible system. 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 functional DNA polymerase III holoenzyme in a single tube using the PURE system. Our results showed that adding 9 DNAs to the PURE system is enough to reconstitute DNA polymerase III holoenzyme, and raised the first direct evidence of that chaperones are not needed to form the complex DNA polymerase III from polypeptides. These are surprising because the classical methods to reconstitute DNA polymerase III holoenzyme need several steps, and co-overexpression of the 9 genes in living cells causes aggregation. Addition of the 13 genes, including initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III holoenzyme, to the reconstituted protein expression 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 reconstitution of an in vitro central dogma cycle.