Role of RuvA in branch migration reactions catalyzed by the RuvA and RuvB proteins of Escherichia coli.
The RuvA and RuvB proteins of Escherichia coli promote ATP-dependent branch migration of Holliday junctions during homologous genetic recombination and DNA repair. In this process, RuvA acts as a specificity factor that targets RuvB, a hexameric ring motor protein, to the junction. Because elevated concentrations of RuvB can promote branch migration in the absence of RuvA, it has been suggested that RuvA acts as a molecular matchmaker. In the studies presented here, we compared the requirements for RuvAB- and RuvB-mediated branch migration reactions and found that reactions catalyzed by RuvB alone were highly sensitive to inhibition by NaCl, temperature, ADP, and ATPgammaS. Our observations indicate that the two reactions occur by distinct mechanisms and support the notion that RuvAB-mediated branch migration is physiologically more relevant than that catalyzed by RuvB. We also show that ongoing RuvAB-mediated branch migration reactions were blocked by the addition of polyclonal antibodies raised against RuvA. The role of RuvA is therefore unlikely to be restricted to RuvB targeting; instead, it is required continually during branch migration. Competition with excess synthetic Holliday junctions, sufficient to sequester released RuvA, failed to cause an immediate block and leads us to suggest that RuvAB promote branch migration by a processive mechanism.
Mitchell
A H
AH
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
West
S C
SC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases