FIG. 8.

In vitro DNA synthesis by Pol III holoenzyme with pRNA synthesized by HT-primase-K241R on 278-nt G4oric template. The synthesis reaction was performed in two stages; first pRNA synthesis and then DNA synthesis. [α-³²P]GTP was used to label the pRNA, and α-³⁵S-dATP was used to label the DNA, as described in Materials and Methods. The synthesis products were analyzed on an 8% polyacrylamide–7 M urea gel, which was dried before autoradiography. Lane 1, size markers of 5′-end γ-³²P-labeled fragments of pBR322 digested with MspI; lane 2, extension with wild-type HT-primase; lane 3, extension with mutant HT-primase-K241R; lane 4, extension with wild-type P47; lane 5, extension with mutant P47-K241R; lane 6, control of DNA synthesis by Pol III without adding primase in the first stage of the reaction.