Functional characterization of wild-type and mutant RecA proteins. (A-C) D-loop formation. The ability to form a D-loop by RecA proteins was determined in the presence of ATP (A), AMP-PNP (B) or ATPγS (C). Reaction samples from the 10 min time point are shown. (D) The ATPase activities of RecA proteins in response to ssDNA. Wild-type or mutant RecA proteins (0.5 mM) were incubated in the presence of 1 mM Mg2+, with or without ΦX174 ssDNA (1 mM nucleotides). ATP hydrolysis was initiated by adding 1 mM ATP (with 0.6 nM [γ-32P]ATP) at 37°C. At different time points, 0.3 μL aliquots were withdrawn and spotted on thin layer chromatography paper to separate [γ-32P]ATP from 32P-labeled inorganic phosphate. All RecA proteins examined here exhibited relatively low ATPase activities (< 0.5 Pi/min/RecA protein) in the absence of ssDNA. Differences between RecA ATPase activities in the presence and absence of ssDNA are shown.