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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System
  • Chromosomes, Bacterial / metabolism*
  • DNA Polymerase III / metabolism
  • DNA Primase / biosynthesis
  • DNA Primase / metabolism
  • DNA Replication*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / biosynthesis
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Gene Regulatory Networks
  • Protein Biosynthesis
  • Protein Subunits / biosynthesis
  • Protein Subunits / genetics
  • Transcription, Genetic

Substances

  • Escherichia coli Proteins
  • Protein Subunits
  • DNA Polymerase III
  • DNA Primase