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.