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

Abstract

Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as chi 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 chi sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of chi-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 chi-specific single-stranded DNA fragment production are observed. Also, under these conditions, chi-specific fragments derived from both the upstream and downstream regions of the DNA strand containing chi and from cleavage of the non-chi-containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD not equal to) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to chi stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, chi itself may be a preferred site for initiation of homologous pairing in this concerted process.