Fig. 6

Measuring the ATPase activity of ParE proteins. a Comparison of basal ATPase rates for wild-type (WT) and representative ParE cavity mutants and stimulation by ParC and DNA. A coupled enzyme assay was used wherein ATPase activity was measured through the change in NADH absorbance at 340 nm^34,35. The reaction mix contained topo IV decatenation buffer, 2 mM ATP and 100 nM ParE and was incubated at 37 °C in the presence or absence of 130 nM ParC and 10 µg pBR322 (final volume of 500 µl). b Bar chart showing the ATPase activity of WT ParE and various single mutants of ParE observed in the absence and presence of DNA and ParC (to form topo IV). Reactions were carried out in triplicate on separate days. Error bars show the standard error. Inclusion of DNA or ParC alone did not stimulate ParE ATPase activity. The ParE D269V mutant showed strong basal ATPase activity that was greatly enhanced by inclusion of ParC and DNA