Substrate specificity of the Escherichia coli RuvC protein. Resolution of three- and four-stranded recombination intermediates.
The specificity of the Escherichia coli RuvC Holliday junction resolvase has been investigated in vitro. RuvC protein cleaves synthetic DNA substrates that model three- or four-stranded recombination intermediates but fails to act upon Y junctions, G/A mismatches, heterologous loop structures, or two-stranded branched junctions. RuvC therefore differs from endonuclease VII of bacteriophage T4 which exhibits broad range specificity. Using related three- and four-stranded synthetic DNA junctions, we show that RuvC cleaves both junctions at the same DNA sequence and requires a region of homology at the junction point. The action of RuvC on three- and four-stranded recombination intermediates made by RecA was also investigated. We found that RuvC fails to resolve three-stranded intermediates in the presence of RecA, although four-stranded intermediates are resolved under the same conditions. However, both three- and four-stranded intermediates are substrates for the nuclease after removal of RecA. We interpret these differences in terms of the contiguity of the RecA nucleoprotein filament which may, under certain conditions, limit access to the Holliday junction resolvase.
Benson
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, United Kingdom.
West
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D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011938:Rec A Recombinases / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D019895:Transposases
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C069806:ruvC protein, E coli; D004247:DNA; D009713:Nucleotidyltransferases; D011938:Rec A Recombinases; D019895:Transposases; D004706:Endodeoxyribonucleases