Figure 4: RecA protein stimulates the DNA-dependent rate of RecN ATP hydrolysis.

ATPase reactions were carried out as described in the Methods section. The ATP hydrolysis measured reflects only that catalysed by RecN protein since RecA K83R is a ATPase-deficient RecA mutant. Linearized pEAW324 plasmid DNA is utilized where indicated. Reactions 1 and 2 are control experiments measuring the amount of ATP hydrolysis over time by RecN protein (2 μM) in the absence (reaction 1) or presence of 50 μM DNA (reaction 2). The order of addition for reactions 3, 4 and 5 are shown (top). The first set (reaction 3, RecN and DNA; reaction 4, RecN and RecA K83R; and reaction 5, RecA K83R and DNA) were incubated with 2.5 mM ATP in buffer N (see Methods) for 20 min before the second addition (reaction 3, RecA K83R; reaction 4, DNA; and reaction 5, RecN). The time of the second addition is indicated by a vertical arrow. The final concentration of RecN, RecA K83R and DNA was 2, 2 and 50 μM, respectively. See Table 1 for steady-state rates of RecN ATP hydrolysis and lag times.