RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.
Bibliographical noteFunding Information:
This work was supported by a National Science Foundation award (PHY-0646550) to T.H. We thank K. Lee, J. Yoo, and S.H. Kim for discussions and experimental help. We thank C. Liu, H. Koh, R. Roy, A. Jain, R. Vafabaksh, R. Zhou, G. Lee, Y. Ishitsuka, P. Cornish, S. Myong, S. Doganay, X. Shi, S.Syed, J. Park, and I. Cisse for advice and comments. We thank M. Schlierf, W. Hwang, M. Spies, and A. Jain for comments on the manuscript.