Figure 4.

DnaE primer extension assays on a long template and DnaE stimulation by DnaN and SSB. Assays were carried out on M13mp18 template (2 nM) with DnaE in the presence and absence of the clamp DnaN (80 nM) and subunits of the clamp loader: DnaX (120 nM), HolB (40 nM), YqeN (40 nM). (a) Primer extension assays at different DnaE concentrations (5, 10, 20, 40, 80, 200, 500 and 1000 nM) primed with DNA and RNA primers, as indicated. Reaction mixtures were incubated at 37°C for 32 min and terminated by the addition of loading dye and incubation at 95°C for 5 min. Quantifications of DnaE-mediated percentage of nascent DNA synthesis is shown underneath each gel. (b,c) Time course reactions using 80 nM DnaE and an RNA primer in the presence or absence of DnaN and/or SSB (1 µM) as indicated. Reactions were incubated at 37°C for the indicated time and terminated by addition of loading dye and incubation at 95°C for 5 min. Reaction products were analysed through 1.5% (w/v) alkaline agarose gels. Lanes labelled M and C show molecular weight markers and a control reaction without polymerases, respectively. All assays were carried out in triplicates and the gels shown here are representative. (d) Quantifications of the primer extension reactions shown in (c).