Activation of RuvC Holliday junction resolvase in vitro.
The Escherichia coli RuvC protein is an endonuclease that resolves Holliday junctions. In vitro, the protein shows efficient structure-specific binding of Holliday junctions, yet the rate of junction resolution is remarkably low. We have mapped the sites of cleavage on a synthetic junction through which a crossover can branch migrate through 26 bp and find that > or = 90% of the junctions were cleaved at one site. This observation of sequence-specific cleavage suggests that inefficient resolution may be due to DNA binding events which occur away from the cleavage site and are therefore non-productive. Holliday junction resolution by RuvC protein can be stimulated by a number of factors including: (i) the presence of Mn2+ (rather than Mg2+) as the divalent metal cofactor, (ii) alkaline pH (< or = 10), and (iii) elevated temperature. These observations may indicate that other proteins are required for efficient RuvC-mediated resolution.
Shah
R
R
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
Bennett
R J
RJ
West
S C
SC
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D017665:Hydroxyl Radical; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics*; D013696:Temperature; D019895:Transposases
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D017665:Hydroxyl Radical; D008345:Manganese; D004247:DNA; D009713:Nucleotidyltransferases; D019895:Transposases; D004706:Endodeoxyribonucleases