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 coli, recognition of the recombination hotspot, chi (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 chi 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 chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. 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 chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.
Arnold
D A
DA
Sections of Microbiology and of Molecular and Cellular Biology, University of California at Davis, CA 95616, USA.
Handa
N
N
Kobayashi
I
I
Kowalczykowski
S C
SC
D000483:Alleles; D000595:Amino Acid Sequence; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009711:Nucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009711:Nucleotides; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli