Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot χ in vitro: Evidence for functional inactivation or loss of the RecD subunit

Genetic recombination in Escherichia coli is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (χ) sites (5'-GCTGGTGG- 3'). Previously, it was shown that χ acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with χ sites can also result in an inactivation of helicase activity of RecBCD. The unwinding of double-stranded DNA-containing χ sites, under conditions of limiting Mg²⁺ ion, results in the reversible inactivation of RecBCD; addition of excess Mg²⁺ to the reaction reactivates all activities of RecBCD. Inactivation is the consequence of a χ-dependent modification of RecBCD that appears to result from an inability of the χ- modified RecBCD to reinitiate unwinding of intact DNA molecules. This characteristic behavior of RecBCD and χ is displayed by the reconstituted RecBC (i.e., without the RecD subunit), except that it is not dependent on χ interaction. This biochemical similarity between the χ-modified RecBCD and RecBC enzymes implies that recognition of χ results in a dissociation or functional inactivation of RecD subunit and lends support to the hypothesis that interaction with χ results in ejection of the RecD subunit.