Abstract
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.
Highlights
Wheneach fixed pair of experiments to allow for small differences the concentrationof recA is increasedat a fixed DNA concen- in the degree of etheno-modification (and fluorescence tration (Fig. L4),the fluorescence increases at a greater rate intensity) between different preparations
We have investigated recA binding to EDNAby the DNA
ATPase activity of recA protein is maximal when recA binds to 3.1 f 0.6 DNA bases, determined from the ATPase rate of saturated DNA and saturated recA
Summary
Dept. of Biological Sciences, Purdue University, The abbreviations used are: ATPyS, adenosine 5’-0-. We have observed that literature procedures for the synthesis of etheno-modified SSDNA, cDNA, often cause degradation of the ssDNA to fragments less than 200 bases long. To minimize this fragmentation of the DNA, we prepared eDNA by incubating 0.3-0.6 mM M13 DNA, 50 mM NaOAc, pH 5.0, 1 mM EDTA, with one-sixth volume freshly distilled chloroacetaldehyde at 45 'C for 90 min. Washing the sample with 20 mM Tris-HC1, pH 7.0, four times in a Centricon 30 was sufficient to remove all of the chloroacetaldehyde.An €280 of 7000 M" cm" was determined for EDNA by a phosphate assay (Bartlett, 1959).
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