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

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.

Figure 1.

Functional DRPs are synthesized in PURE system. Proteins synthesized in the PURE system were used without purification for assays. SSB was firstly added to inhibit formation of heretoduplex due to T7 RNA polymerase transcriptional activity in the PURE system. (A) A representative figure of DNA Pol III HE. (B) A schematic representation of G4 replication assay. (C) Pol III HE expression at temperatures. The 9 genes that comprise Pol III HE were mixed with the PURE system at three temperatures, 37, 30 and 25°C. (D and F): De novo synthesized DnaG and Pol III HE could be replaced with purified enzymes. Pol III HE and DnaG were required for DNA replication of G4 ssDNA. Purified enzymes were used at 1.0-, 0.33-, 0.11- and 0-fold the amount indicated in the ‘Materials and Methods’ section. The de novo synthesized proteins were 3, 1 and 0.3 μl of the PURE system reaction mixture in 10 μl of total volume. As a negative control, 1 μl of the PURE system mixture without protein expression was used. Arrows indicated the corresponding bands of non-replicated DNA (ssDNA) and replicated dsDNA. (E) Essentiality of the 9 genes for Pol III HE expression was assessed by replication of G4 ssDNA. Positive: purified Pol III HE, Negative: no DNA polymerase, All: all of the 9 genes were synthesized using the PURE system. Genes omitted: each gene was omitted from the 9 gene mixture. The hol genes indicate the mixture of genes, holA, holB, holC, holD and holE. (G) A schematic representation of G4 replication assay. (H): Replacement assay of de novo synthesized DnaA, DnaB and DnaC with purified enzymes. Adding DnaA, DnaB, DnaC, SSB, IHF and gyrase alters the topology of DNA that have a chromosomal DNA replication origin (oriC). The topological differences were separated by low voltage gel electrophoresis. Purified enzymes were used at 1.0-, 0.33-, 0.11- and 0-fold of the amount indicated in ‘Materials and Methods’ section. The DnaA, DnaB and DnaC (DnaC was diluted into 1/16-fold with buffers) synthesized using the PURE system were used at 2.0, 0.67 and 0.22 μl in a 10 μl total volume. As a negative control, 2 μl of the PURE system mixture without protein expression was used. Arrows indicated the corresponding bands of form I and form I*.

Figure 2.

A mixture of DRPs that are expressed by the PURE system works in concert to replicate chromosomal DNA. (A) A schematic representation of A-site ssDNA replication assay. (B) DRPs were synthesized by using the PURE system in different tubes, and then mixed to examine A-site ssDNA replication. Green characters indicate purified proteins were used, and magenta characters indicate that de novo proteins synthesized through the PURE system were used. All: DnaA, DnaB, DnaC, DnaG and Pol III HE were used for DNA replication. None: no DRPs were added. Component omitted: each protein described above lanes was not added the reaction mixtures. All (PURE system mixtures): 1.6, 1.5, 0.12, 1.5 and 3.0 μl of DnaA, DnaB, DnaC, DnaG, Pol III HE synthesized in PURE system were mixed in the 10-μL final mixture. FtsZ: PURE system expressing ftsZ gene was used as a negative control because solution from PURE system mixtures comprises 80% of the volume of total mixtures. SSB was firstly added to inhibit formation of heretoduplex due to T7 RNA polymerase transcriptional activity in the PURE system.

Figure 3.

Autonomous DNA replication by expression of DRPs using PURE system in a single tube. (A) A schematic diagram of a coupling reaction in which protein expression induces DNA replication. The 10 genes for G4 ssDNA replication and the additional 3 genes for A-site ssDNA replication are indicated by blue and red characters, respectively. (B, C): The result of the coupling reaction, in which PURE system synthesizes DRPs, and DRPs replicated DNA in a single tube. (B, C): SSB was added before DRPs synthesis by the PURE system. (B) ‘negative’, ‘10 genes’ and ‘ftsZ’ indicate no DNA for protein expression, the 10 genes described by blue character in Fig 3A, and ftsZ gene as a negative control were added, respectively. (C) ‘negative’, ‘13 genes’ and ‘ftsZ’ indicate no DNA, the 13 genes described in Fig 3A and ftsZ gene as a negative control were added, respectively. In the case of the 12 genes, dnaC gene was omitted from the 13 genes. (D): Plaque-forming assay for evaluating biological activity of the replicated DNA by the coupling reaction in a single tube. DNA purified through phenol/CHCl₃ and RNaseA treatment was used. Relative units indicate the rate of plaque numbers, when the plaque number of the replicated DNA by purified enzymes was determined as 100%. The 100% indicates 615 plaques for G4 ssDNA and 1434 plaques for A-site ssDNA.

Figure 4.

Two-round flow of the reconstituted central dogma system revealed by an artificial genetic circuit. (A) Schematic representation of the artificial genetic circuit to detect two-round flow of the reconstituted central dogma. An ssDNA that has T7-GFP sequence and A-site origin (T7GFP–A-site ssDNA) was used. Only the replicated form of T7GFP–A-site DNA is able to express GFP. In the artificial circuit, the PURE system with the 13 gene synthesizes DRPs. DRPs replicated T7GFP–A-site ssDNA, and the replicated DNA provides information for GFP expression. Thus, GFP expression indicates the reconstituted central dogma works two rounds. (B) Experimental results of two-round central dogma assay in a single tube. SSB was added before DRPs synthesis by PURE system. The upper panel shows DNA replication of T7GFP–A-site ssDNA. In the case of T7GFP–A-site ssDNA under our conditions, migration of the replicated DNA is faster than the not-replicated DNA. Arrows indicated the corresponding bands of the not-replicated DNA (ssDNA) and of the replicated dsDNA. The lower panel shows GFP expression through the artificial genetic circuit. The ‘12 genes’ indicates omission of the dnaC gene from the 13 genes. All samples used in this experiment contain T7GFP–A-site ssDNA. The case of the ‘13 genes’, 127 nM of GFP was expressed.