Analysis of two distinct single-stranded DNA binding sites on the recA nucleoprotein filament.
The binding stoichiometry of Escherichia coli recA protein to single-stranded DNA (ssDNA) determined by two separate assays differs by a factor of 2.2-2.4. Using the fluorescence of etheno-DNA (epsilon DNA), a chemically modified ssDNA, the stoichiometry was found to be 7.0 +/- 0.6 bases/recA protein monomer in a nucleo-protein filament. Using a competition assay, a similar stoichiometry, 7.5 bases/recA, is found for unmodified poly(dT). Using the DNA-dependent ATPase of recA, which monitors bound protein rather than bound DNA, we find that each recA monomer 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 that there are two DNA binding sites with differential effects on ATPase activation. When recA protein is mixed with ssDNA at a ratio of 7 bases/recA or greater, the complex that forms contains 7 bases/recA and acts as a kinetic trap for the ssDNA. Upon further addition of recA protein, 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 bases/recA or less) the ATPase activity is twice as high. Analysis of the 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 ATPase activity of recA is completely active in this complex. A second strand of DNA can then be bound to this filament yielding a final stoichiometry of approximately 7 bases/protein monomer. The presence of this second strand neither enhances nor inhibits ATP hydrolysis. This ternary complex may mimic the structures formed by recA in searching for homologous DNA sequences and/or in the strand exchange reaction.
Zlotnick
A
A
DuPont Merck Pharmaceutical Company, Experimental Station, Wilmington, Delaware 19880-0328.
Mitchell
R S
RS
Steed
R K
RK
Brenner
S L
SL
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001665:Binding Sites; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D011938:Rec A Recombinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases