DNase I Footprinting Analysis of RecA Protein Polymerized on DNA during Strand Exchange Reaction between a Gapped Circle and a Linear Duplex1

RecA protein mediates homologous pairing and strand exchange reactions between a circular duplex with a single strand gap and a linear duplex. We have used the DNase I footprinting method to analyze processes involving four strands during these reactions. We asked how the length of DNA protected by RecA protein changes as these reactions proceed. We compared two kinds of gapped DNAs. We found that RecA protein polymerizes rapidly in the forward direction (5' to 3' with respect to the single strand). We found, however, that polymerization in the reverse direction was more prominent with a duplex carrying a longer gap than one carrying a shorter gap. DNase I footprints showing protection by RecA protein were obtained only at limited nuclease concentrations, which in turn depended on the position of the end label and the stage of the strand exchange reaction. As judged by the concentrations of DNase I good for footprinting, the extent of protection by RecA protein was greatest for (+) single-stranded DNA at its first binding site, next highest for heteroduplex containing this (+) strand, and least for the gapped homoduplex DNA. These differences in DNase I sensitivity can be explained in terms of differences in the accessibility of various strands on the basis of a three-dimensional model for the strand exchange reaction.