Significance Homologous recombination (HR) is a major pathway for repair of double-stranded DNA break. The RecA proteins form a helical nucleofilament on single-stranded DNA (ssDNA) with a periodicity of ∼18 bases, which searches a donor DNA that is homologous to the ssDNA. The key point in HR is how the filament senses the sequence of the donor DNA. We designed a series of mismatch-containing donor DNAs to study HR and found that the strand exchange was blocked remotely by the mismatches. Our data suggest that the strand exchange progresses iteratively with the sequences being checked successively in ∼9-bp steps in the frontier of the extending synapsis, followed by a transitional segment that leads the post-strand-exchanged duplex.