The reduced levels of χ recognition exhibited by the RecBC¹⁰⁰⁴D enzyme reflect its recombination defect in vivo

Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme and is stimulated by an 8-nucleotide element known as Chi (χ). We present a detailed biochemical characterization of a mutant RecBCD enzyme, designated RecBC¹⁰⁰⁴D, that displays a reduced level of χ site recognition. Initially characterized genetically as unable to respond to the χ sequence, we provide evidence to indicate that the ability of this mutant enzyme to respond to χ is reduced rather than lost; the mutant displays about 20-fold lower χ recognition than wild-type RecBCD enzyme. Although this enzyme exhibits wild-type levels of double-stranded DNA exonuclease, helicase, and ATPase activity, its ability to degrade single-stranded DNA is enhanced 2-3-fold. The data presented here suggest that the reduced recombination proficiency of the recBC¹⁰⁰⁴D strain observed in vivo results from a basal level of modification of the RecBC¹⁰⁰⁴D enzyme at both χ-specific, as well as nonspecific, DNA sequences.