Figure 3.
DnaE and DnaEpol- activity assays on short templates. DNA primer (a,b) or RNA primer (c) extension assays using short radiolabelled deoxyribonucleotide or ribonucleotide (15 nt) annealed onto longer oligonucleotide template (110 nt) for the detection of DnaE activity. These were performed using protein concentration titration assays (5, 10, 20, 40, 80, 200, 500 and 1000 nM) with either DnaEWT (a,c) or DnaEpol- mutant protein (b). The reaction samples were incubated at 37°C for 15 min, resolved by electrophoresis on 15% (v/v) urea gel PAG, the results were analysed using molecular imager and associated software (Bio-Rad) and the percentage of the assay products was plotted using GraphPad Prism 6. Bars represent mean values with standard errors of at least three independent protein samples. (d–f) EMSAs showing DnaE binding to different radiolabelled nucleotide substrates. The reaction mixture of buffer and substrate (0.66 nM) was pre-incubated at 37°C for 5 min before addition of DnaE at different concentrations, as indicated, and further incubation at 37°C for 5 min. Samples were mixed with loading buffer and analysed through 5% (v/v) native polyacrylamide gels. Lanes labelled 0 show the control radiolabelled substrate on its own. Asterisks indicate the radiolabel position at the 5′-P end.