Topological testing of the mechanism of homology search promoted by RecA protein

To initiate homologous recombination, sequence similarity between two DNA m olecules must be searched for and homology recognized. How the search for a nd recognition of homology occurs remains unproven. We have examined the in fluences of DNA topology and the polarity of RecA-single-stranded (ss)DNA f ilaments on the formation of synaptic complexes promoted by RecA, Using two complementary methods and various ssDNA and duplex DNA molecules as substr ates, we demonstrate that topological constraints on a small circular RecA- ssDNA filament prevent it from interwinding with its duplex DNA target at t he homologous region, We were unable to detect homologous pairing between a circular RecA-ssDNA filament and its relaxed or supercoiled circular duple x DNA targets. However, the formation of synaptic complexes between an inva ding linear RecA-ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a tri pler structure formed by non-Watson-Crick hydrogen bonding is unlikely to b e an intermediate in homology searching promoted by RecA. Rather, a model i n which RecA-mediated homology searching requires unwinding of the duplex D NA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA-ssDNA does not affect its abili ty to pair and interwind with its circular target duplex DNA.