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. 2013 Jun 3;41(14):7176–7183. doi: 10.1093/nar/gkt489

Figure 1.

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*.