Figure 5: The stimulation of RecN ATPase by RecA protein under D-loop assay conditions is not homology-dependent.

(a) Schematic of reaction assembly used to monitor RecN ATPase during RecA-dependent D-loop formation. RecA K83R (3.4 μM where indicated) was incubated with probe DNA (see Fig. 2 legend) for 10 min before the addition of 3 mM ATP and RecN (1 μM where indicated). Target DNA (see Fig. 2 legend) was added 10 min later. For each reaction described, components omitted from reactions were compensated for by protein storage buffers or TE, in the case of DNA. All reactions were carried out under buffer A conditions and followed the reaction scheme shown. ATP hydrolysis was measured after the addition of ATP. (b) Controls measuring RecN ATP hydrolysis in the absence of RecA K83R are shown with 10 μM probe DNA and no target DNA (reaction 1), no probe DNA and 10 μM target DNA (reaction 2), and 10 μM probe DNA plus 10 μM target DNA (reaction 3). Reaction 4: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA in the absence of added target DNA. Reaction 5: RecN ATP hydrolysis when RecA K83R protein was incubated in the absence of probe DNA followed by 10 μM target DNA. Reaction 6: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA followed by 10 μM target DNA. Reaction 7: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA followed by 10 μM non-homologous, supercoiled RF1 φX174 DNA. See Table 2 for steady-state RecN ATP hydrolysis rates.