ANALYSIS OF 2 DISTINCT SINGLE-STRANDED-DNA BINDING-SITES ON THE RECA NUCLEOPROTEIN FILAMENT

The binding stoichiometry of Escherichia coli recA protein to single-s tranded DNA (ssDNA) determined by two separate assays differs by a fac tor of 2.2-2.4. Using the fluorescence of etheno-DNA (epsilonDNA), a c hemically modified ssDNA, the stoichiometry was found to be 7.0 +/- 0. 6 bases/recA protein monomer in a nucleoprotein filament. Using a comp etition assay, a similar stoichiometry, 7.5 bases/recA, is found for u nmodified poly(dT). Using the DNA-dependent ATPase of recA, which moni tors bound protein rather than bound DNA, we find that each recA monom er needs to bind only 3.1 +/- 0.5 bases to fully activate the ATPase. The difference in site size determined by the two assays indicates tha t there are two DNA binding sites with differential effects on ATPase activation. When recA protein is mixed with ssDNA at a ratio of 7 base s/recA or greater, the complex that forms contains 7 bases/recA and ac ts as a kinetic trap for the ssDNA. Upon further addition of recA prot ein, no additional ATPase activity is observed. If, on the other hand, the ssDNA is initially mixed with excess recA (at a ratio of 3-3.5 ba ses/recA or less) the ATPase activity is twice as high. Analysis of th e binding curves suggests that the first DNA strand binds recA to form a filament with a stoichiometry of 3-3.5 bases/protein monomer. The A TPase activity of recA is completely active in this complex. A second strand of DNA can then be bound to this filament yielding a final stoi chiometry of -7 bases/protein monomer. The presence of this second str and neither enhances nor inhibits ATP hydrolysis. This ternary complex may mimic the structures formed by recA in searching for homologous D NA sequences and/or in the strand exchange reaction.