Targeting Holliday junctions by the RecG branch migration protein of Escherichia coli.
The RecG protein of Escherichia coli is a junction-specific DNA helicase that drives branch migration of Holliday intermediates in genetic recombination and DNA repair. The reaction was investigated using synthetic X-junctions. RecG dissociates X-junctions to flayed duplex products, although DNA unwinding of the heterologous arms is limited to </=30 base pairs. Junction unwinding requires Mg2+ and the hydrolysis of ATP. X-junction DNA stimulates the ATPase activity of RecG. ATPase activity is also stimulated by linear duplex DNA, although to a lesser extent than by X-DNA, but not by linear single-stranded DNA. In situ 1,10-phenanthroline-copper footprinting shows that RecG binds to the strand cross-over point at the center of the X-junction. Substrate recognition by RecG was investigated using DNAs that represented the various component parts of an X-junction. The minimal DNA structure that RecG forms a stable complex with is a flayed duplex, suggesting that this is the critical feature for junction recognition by RecG. Junction binding and unwinding also depend critically on the concentration of free Mg2+, excess free cation dramatically inhibiting both processes. These inhibitory effects are not mediated specifically by Mg2+; e.g. both Ca2+ and hexamminecobalt(III) chloride also inhibit X-junction binding and unwinding by RecG. The relative abilities of these cations to inhibit RecG-junction binding is correlated with their respective abilities to stack X-junction DNA. From this we conclude that RecG is unable to bind or binds very poorly to fully stacked X-junctions.
Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. whitby@bioch.ox.ac.uk
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D008274:Magnesium / Q000494:pharmacology; D010618:Phenanthrolines / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*
D001426:Bacterial Proteins; D002712:Chlorides; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010618:Phenanthrolines; C007936:hexaminecobalt (III) trichloride; C071446:RecG protein, E coli; D003035:Cobalt; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; D008274:Magnesium; C025205:1,10-phenanthroline