Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA

The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I, This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the p resence of adenosine 5'-O-(thiotriphosphate) cofactor using fluorescence an isotropy, The RecA protein carried out DNA strand exchange with a 5'-fluore scein-labeled 32-mer oligonucleotide. The anisotropy signal was shown to me asure oligonucleotide binding to RecA, and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described b y a simple two-state equilibrium, K = 4.5 +/- 1.5 x 10(5) M-1 (37 degreesC, 150 mM NaCl, pH 7,4). The reaction was enthalpy-driven and entropy-opposed . It depended on salt concentration and was sensitive to the type of monova lent anion, suggesting that anion-dependent protein conformations contribut e to ssDNA binding at site II.