The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts.
The Escherichia coli RuvC protein resolves DNA intermediates produced during genetic recombination. In vitro, RuvC binds specifically to Holliday junctions and resolves them by the introduction of nicks into two strands of like polarity. In contrast to junction recognition, which occurs without regard for DNA sequence, resolution occurs preferentially at sequences that exhibit the consensus 5'-(A/T)TT/(G/C)-3' (where / indicates the site of incision). Synthetic Holliday junctions containing modified cleavage sequences have been used to investigate the mechanism of cleavage. The results indicate that specific DNA sequences are required for the correct docking of DNA into the two active sites of the RuvC dimer. In addition, using chemically modified oligonucleotides to introduce a hydrolysis-resistant 3'-S-phosphorothiolate linkage at the cleavage site, it was found that, as long as the sequence requirements are fulfilled, the two incisions could be uncoupled from each other. These results indicate that RuvC protein resolves Holliday junctions by a mechanism similar to that exhibited by certain restriction enzymes.
Shah
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, UK.
Cosstick
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West
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SC
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C069806:ruvC protein, E coli; D004706:Endodeoxyribonucleases