The search for DNA homology does not limit stable homologous pairing promoted by RecA protein

Abstract

The basic molecular mechanisms that govern the search for DNA homology and subsequent homologous pairing during genetic recombination are not understood. RecA is the central homologous recombination protein of Escherichia coli; because several RecA homologues have been identified in eukaryotic cells, it is likely that the mechanisms employed by RecA are conserved throughout evolution. Analysis of the kinetics of the homologous search and pairing reactions catalyzed by RecA should therefore provide insights of general relevance into the mechanisms by which macromolecules locate, and interact with, specific DNA targets. RecA forms three-stranded synaptic complexes with a single-stranded oligonucleotide and a homologous region in duplex DNA. The kinetics of this initial pairing reaction were characterized using duplex DNA molecules of various concentrations and complexities containing a single target site, as well as various concentrations of homologous single-stranded oligonucleotides. The formation of the synaptic complex follows apparent second-order reaction kinetics with a rate proportional to the concentrations of both the homologous single-stranded oligonucleotide and the target sites within the duplex DNA. The reaction rate is independent of the complexity of duplex DNA in the reaction. We propose a kinetic scheme in which the RecA-single-stranded DNA filament interacts with duplex DNA and locates its target in a relatively fast reaction. We also suggest that complex conformational changes occur during the subsequent rate-limiting step. We conclude that, during the formation of synaptic complexes by RecA, the search for homology is not rate-limiting, and that the iteration frequency of the search is around 10(2)-10(3) s-1. This value agrees well with what has been calculated as the minimum number for such a frequency in genome-wide searches, and limits the possible structures involved in the search for homology to those involving very soft (low energy) interactions. Furthermore, from the order of the reaction at the DNA concentrations found in eukaryotic nuclei, and the rate constant of the overall reaction, we predict that the search for homology is also not the rate-limiting step in the genome-wide searches implicated in meiosis and in gene targeting.