Evidence for a four-strand exchange catalyzed by the RecA protein

Strand exchange between two duplexes is usually initiated as a three-strand event that requires the presence of a single-stranded overhang or gap in o ne of the two molecules. Here we show that the RecA protein can catalyze a four-strand exchange. Specifically, it can recombine short hairpin substrat es with homologous stems provided that one of the hairpins possesses a chim eric DNA/RNA backbone. This four-strand exchange reaction goes to completio n in the presence of ATP gammaS and releases a stable heteroduplex upon rem oval of the RecA protein. Under identical conditions, strand exchange betwe en two DNA hairpins is incomplete and generates a nascent heteroduplex that rapidly dissociates when the RecA protein is denatured. Since presynaptic filament formation does not appear to melt either type of hairpin, we propo se that exchange occurs between homologously aligned duplexes that are exte nded and unwound within a RecA filament. The first reaction provides a mech anism for gene targeting by chimeric double-hairpin oligonucleotides while the second reaction explains the ability of the RecA protein to transiently align double-stranded DNA molecules.