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Nucleic Acids Res. 2013 Aug;41(14):7176-83. doi: 10.1093/nar/gkt489. Epub 2013 Jun 3.

Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system.

Author information

1
Department of Bioengineering and Robotics, Tohoku University, 6-6-01, Aramakiaza-aoba, Aoba-ku, Sendai, Miyagi, 980-8579, Japan. fujiwara@molbot.mech.tohoku.ac.jp

Abstract

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.

PMID:
23737447
PMCID:
PMC3737561
DOI:
10.1093/nar/gkt489
[Indexed for MEDLINE]
Free PMC Article

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