PMID:10884344
| Citation |
Arnold, DA, Handa, N, Kobayashi, I and Kowalczykowski, SC (2000) A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi. J. Mol. Biol. 300:469-79 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/jmbi.2000.3861 |
| Keywords |
Alleles; Amino Acid Sequence; Base Pairing/genetics; Base Sequence; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; DNA, Single-Stranded/genetics; DNA, Single-Stranded/metabolism; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Exodeoxyribonuclease V; Exodeoxyribonucleases/chemistry; Exodeoxyribonucleases/genetics; Exodeoxyribonucleases/metabolism; Genetic Variation/genetics; Molecular Sequence Data; Mutation/genetics; Nucleotides/genetics; Plasmids/genetics; Plasmids/metabolism; Rec A Recombinases/metabolism; Recombination, Genetic/genetics; Regulatory Sequences, Nucleic Acid/genetics; Substrate Specificity |
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