A novel, 11 nucleotide variant of χ, χ*: One of a class of sequences defining the Escherichia coli recombination hotspot χ

In wild-type Escherichia coli, recognition of the recombination hotspot, χ (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical χ sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of χ, termed χ* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the χ* sequence is recognized more efficiently by the RecBC¹⁰⁰⁴D enzyme than is the wild-type χ. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at χ* than at χ. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of χ-recognition. In addition, we show that this novel, 11 nucleotide χ* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical χ constitute a class of sequences that regulate the recombination function of RecBCD enzyme. (C) 2000 Academic Press.