Real-Time Observation of Strand Exchange Reaction with High Spatiotemporal Resolution

Abstract

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 basepair and milliseconds resolution which can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggests 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.

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

“…Rapid kinetics data support the idea that the RecA-ssDNA filament binds to a deformed conformation of the duplex in which the sequence may be sampled via the formation of Watson-Crick base pairs (Lee et al 2006). The propagation of heteroduplex formation occurs in three-base-pair increments (Ragunathan et al 2011), consistent with the organization of the ssDNA observed in the RecA-ssDNA-ADPAlF 4 crystal structure (Chen et al 2008).…”

“…Cite this article as Cold Spring Harb Perspect Biol 2015;7:a016444 the idea that the filament senses both WatsonCrick base pairing and DNA conformation in the recognition process, a mechanism referred to as "conformational proofreading" (Lee et al 2006;Takahashi et al 2007; see comments in Rambo et al 2010;Savir and Tlusty 2010;Ragunathan et al 2011). The atomic structure of the RecA-ssDNA filament, in its active, ATP-bound form, reveals that the ssDNA exists in a deformed conformation, with the sugar-phosphate backbone extended between trinucleotide units, which are held in a B-like conformation (Chen et al 2008; see comments by Kowalczykowski 2008).…”

Base triplet stepping by the Rad51/RecA family of recombinases

“…We have also reported that strand exchange by Saccharomyces cerevisiae Rad51 (ScRad51) occurs in 3-nt steps, presumably reflecting the base triplet organization of RS-DNA (Fig. 1, A and B) (11), and 3-nt stepping has also been proposed for bacterial RecA (12). Here, we sought to determine the underlying principles that contribute to base triplet stepping and establish how these principles influence the mechanism and fidelity of DNA recombination.…”

A change of view: homologous recombination at single-molecule resolution

“…Nevertheless, an assay that combines TIRFM and smFRET has been used to visualize the capture of homologous dsDNA fragments by a RecA presynaptic complex anchored to a slide surface 112 . Using this approach, strand exchange led to a high FRET signal between a Cy5 acceptor positioned on the presynaptic complex and a Cy3 donor placed on the incoming dsDNA.…”