Complement-stabilized D-loop: RecA-catalyzed Stable Pairing of Linear DNA Molecules at Internal Sites

Abstract

The RecA protein (RecA) of Escherichia coli has the ability to pair a single-stranded DNA to a homologous sequence in a duplex DNA without requiring denaturation of the duplex. This ability has stimulated interest in the use of RecA for targeting to genomic DNA. However, because pairing generally requires that the double-stranded DNA either have a homologous end or be negatively supercoiled, the application of RecA to targeting has been very limited. Here, we show that if the sequence complementary to the probe is also included in the reaction, RecA can pair the two single strands to sites distant from any ends on linear DNA. The resulting structure, termed a complement-stabilized D-loop (csD-loop), is cleavable by restriction endonucleases and, upon removal of RecA, remains stable to temperatures up to the t m of the double-stranded probe. These results indicate that the csD-loop probably consists of two side-by-side Watson-Crick duplexes, much like a replication bubble. This novel reaction of RecA may be useful in gene mapping and isolation, as well in sequence-specific cleavage of genomic DNA, and might have functions in vivo.