Construction and evaluation of a kinetic scheme for RecA‐mediated DNA strand exchange

The Escherichia coli RecA protein is the prototype of a class of proteins playing a central role in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding its recombinational activities. Toward a molecular‐level understanding of RecA‐mediated DNA strand exchange, we explored its mechanism using oligonucleotide substrates and the intrinsic fluorescence of 6‐methylisoxanthopterin (6MI). Steady‐ and presteady‐state spectrofluorometric data demonstrate that the reaction proceeds via a sequential four‐step mechanism comprising a rapid, bimolecular association step followed by three slower unimolecular steps. Previous authors have proposed multistep mechanisms involving two or three steps. Careful analysis of the differences among the experimental systems revealed a previously undiscovered intermediate (N 1 ) whose formation may be crucial in the kinetic discrimination of homologous and heterologous sequences. This observation has important implications for probing the fastest events in DNA strand exchange using 6MI to further elucidate the molecular mechanisms of recombination and recombinational repair. © 2006 Wiley Periodicals, Inc. Biopolymers 81: 473–496, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com