Figure 5.

DnaG differentially modulates DNA synthesis mediated by PolC or by DnaE. (A) Scheme of the synthetic nicked mini-circle substrate used in these assays; leading strand synthesis is primed by the pre-existing 3’-OH DNA end, and with this substrate σ-type concatemeric DNA replication is obtained. (B) DnaG inhibits leading strand synthesis catalyzed by DnaE. DNA replication reactions were performed with the synthetic nicked mini-circle as DNA template in the presence of [α-³²P]dCTP or [α-³²P]dGTP to visualize leading or lagging strand synthesis respectively. In addition to DnaE, reactions had G38P, G39P, G40P, τ complex, β and 100 nM SsbA as the SSB protein. As indicated, reactions were performed in the absence or presence of increasing DnaG concentrations (8, 16 and 32 nM), and were incubated for 40 min at 37 °C. (C) DnaG stimulates leading strand synthesis catalyzed by a complete replisome. Increasing concentrations of DnaG (0, 8, 16, and 32 nM) were added to the full SPP1 replisome (PolC plus DnaE and all replisome components). Reactions contained [α-³²P]dCTP or [α-³²P]dGTP to visualize leading or lagging strand synthesis and were incubated for 2 min at 37°C. (D) DnaG stimulates leading strand synthesis catalyzed by PolC. Reactions were performed similarly but here [α-³²P]dCTP was used, and PolC was the sole DNA polymerase present. ld: leading strand, lg: lagging strand; M: DNA ladder used, HindIII labelled λ DNA.