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J Biol Chem, Vol. 273, Issue 31, 19729-19739, July 31, 1998

Targeting Holliday Junctions by the RecG Branch Migration Protein of Escherichia coli

Matthew C. Whitby and Robert G. Lloyd^¶

From the  Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom and the ^¶ Department of Genetics, University of Nottingham, Queens Medical Center, Nottingham NG7 2UH, United Kingdom

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.

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