THE SEARCH FOR DNA HOMOLOGY DOES NOT LIMIT STABLE HOMOLOGOUS PAIRING PROMOTED BY RECA PROTEIN

Background: The basic molecular mechanisms that govern the search for DNA homology and subsequent homologous pairing during genetic recombin ation are not understood. RecA is the central homologous recombination protein of Escherichia call; because several RecA homologues have bee n identified in eukaryotic cells, it is likely that the mechanisms emp loyed by RecA are conserved throughout evolution. Analysis of the kine tics of the homologous search and pairing reactions catalyzed by RecA should therefore provide insights of general relevance into the mechan isms by which macromolecules locate, and interact with, specific DNA t argets. Results: RecA forms three-stranded synaptic complexes with a s ingle-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 conta ining a single target site, as well as various concentrations of homol ogous single-stranded oligonucleotides. The formation of the synaptic complex follows apparent second-order reaction kinetics with a rate pr oportional to the concentrations of both the homologous single-strande d oligonucleotide and the target sites within the duplex DNA. The reac tion rate is independent of the complexity of duplex DNA in the reacti on. We propose a kinetic scheme in which the RecA-single-stranded DNA filament interacts with duplex DNA and locates its target in a relativ ely fast reaction. We also suggest that complex conformational changes occur during the subsequent rate-limiting step. Conclusions: We concl ude that, during the formation of synaptic complexes by RecA, the sear ch 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 i n genome-wide searches, and limits the possible structures involved in the search for homology to those involving very soft (low energy) int eractions. Furthermore, from the order of the reaction at the DNA conc entrations found in eukaryotic nuclei, and the rate constant of the ov erall reaction, we predict that the search for homology is also not th e rate-limiting step in the genome-wide searches implicated in meiosis and in gene targeting.