Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins. I. Comparison of RuvAB- and RuvB-mediated reactions.
The Escherichia coli RuvA and RuvB proteins mediate the branch migration of Holliday junctions in vitro. In the presence of stoichiometric amounts of RuvB (1 RuvB dimer/12 nucleotides), branch migration can occur without need for RuvA. However, RuvA is required when the RuvB concentration is reduced 4-fold or more. Under optimal conditions, we found the minimal protein requirement to be 1 RuvB dimer per 500-1100 nucleotides and 1 RuvA tetramer per 600-1200 nucleotides. To determine the roles of RuvA and RuvB in branch migration, we compared branch migration reactions mediated by RuvB only and by RuvA and RuvB. The time courses of the two reactions were similar, and both required ATP and Mg2+. However, RuvB-mediated branch migration occurred at lower ATP concentrations (> or = 200 microM) and higher Mg2+ concentrations (> or = 10 mM MgCl2) than the reaction mediated by RuvA and RuvB (> or = 1 mM ATP, > or = 5 mM MgCl2). The Mg2+ requirement for RuvB-mediated branch migration reflects the Mg2+ requirement of RuvB for DNA binding (Müller, B., Tsaneva, I.R., and West, S. C. (1993) J. Biol. Chem. 268, 17185-17189) and can be overcome by addition of RuvA. These results indicate that RuvA protein facilitates the interaction of RuvB with DNA.
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, United Kingdom.
B
B
Müller
I R
IR
Tsaneva
S C
SC
West
D001426:Bacterial Proteins / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases