Role of the Escherichia coli recombination hotspot, χ, in RecABCD-dependent homologous pairing

Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as χ sites, 5′-GCTGGTGG-3′. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a χ sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of χ-specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg²⁺ concentration, joint molecule formation does not occur, even though DNA unwinding and χ-specific single-stranded DNA fragment production are observed. Also, under these conditions, χ-specific fragments derived from both the upstream and downstream regions of the DNA strand containing χ and from cleavage of the non-χ-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD^‡) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to χ stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, χ itself may be a preferred site for initiation of homologous pairing in this concerted process.