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

In wild-type Escherichia coil, recognition of the recombination hotspot, A( 5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombinatio n. However, in the recC* class of RecBCD mutants, stimulation of recombinat ion by the canonical chi sequence is not detectable, but the levels of homo logous 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 chi, termed chi* (5'-GCTGGTGCTCG-3'). H ere, we establish that, in vitro, the chi* sequence is recognized more effi ciently by the RecBC(1004)D enzyme than is the wild-type chi. This is manif est by both a greater modification of nuclease activity and a higher stimul ation of RecA protein-mediated joint molecule formation at chi* than at chi Sequencing of the recC1004 gene revealed that it contains a frameshift mut ation, which results in a replacement of nine of the wild-type amino acid r esidues by eight in the mutant protein, and defines a locus that is importa nt for the specificity of chi-recognition. Ln addition, we show that this n ovel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzym e, supporting the notion that variants of the canonical chi, constitute a c lass of sequences that regulate the recombination function of RecBCD enzyme . (C) 2000 Academic Press.