FIGURE 4.
RecFOR proteins load RecA protein onto gapped DNA in the presence of a large excess single-stranded DNA. A, DNA strand exchange was measured using M13 ssDNA (5000 nm) and φX174 gDNA (500, 50, or 5 nm). SSB protein and RecFOR proteins were incubated for 10 min with the DNA. 32P-Labeled φX dsDNA was then added, and the reaction was started by adding RecA protein. After 90 min, the sample was taken for analysis by agarose gel electrophoresis and autoradiography. The RecA concentrations were 2, 0.2, 0.02, 0.002, or 0 μm in each set (from left to right); “c” represents the control sample taken at 0 min in the presence of 2 μm RecA protein. Arrows indicate the positions of φX174 DNA product and 35-mer dsDNA. The faint bands at the top of gel correspond to a small amount of contaminant in φX174 DNA substrate with a higher molecular weight. B, graphs showing the yield of DNA strand exchange product, normalized for the amount of gDNA present, were determined from agarose gel electrophoresis and autoradiography for the two RecA protein concentrations, 2 and 0.2 μm. The graphs show the results from 2 or 3 independent experiments, and error bars show standard error.