The steady-state kinetic parameters of the ATPase activity of the homohexameric DNA helicase RepA and the binding of the fluorescent analogue epsilon ADP to RepA have been studied. ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves in both the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA-stimulated ATPase activity. Fluorescence titrations show that, at 25 degrees C and in the absence of DNA, the binding of epsilon ADP to RepA is biphasic with three high (K-1 = 1.54 x 10(6) M-1) and three low (K-2 = 4.71 x 10(4) M-1) affinity binding sites differing by 30-40-fold in binding constants. In the absence of cofactors, RepA melts cooperatively at T-m= 65.8 +/- 0.1 degrees C and is more stable in the presence of ATP gamma S, T-m = 68.1 +/- 0.2 degrees C (Delta Delta G 0.95 kcal/mol), than in the presence of ADP, T-m = 66.5 +/- 0.1 degrees C (Delta Delta G 0.29 kcal/mol), indicating that the additional phosphate group in ATP gamma S has a significant influence on RepA structure. A model is proposed in which individual subunits of RepA sequentially and cooperatively perform a multistep ATP hydrolytic cycle.