TARGETING HOLLIDAY JUNCTIONS BY THE RECG BRANCH MIGRATION PROTEIN OF ESCHERICHIA-COLI

The RecG protein of Escherichia coli is a junction-specific DNA helica se that drives branch migration of Holliday intermediates in genetic r ecombination and DNA repair. The reaction was investigated using synth etic X-junctions. RecG dissociates X-junctions to flayed duplex produc ts, although DNA unwinding of the heterologous arms is limited to less than or equal to 30 base pairs. Junction unwinding requires Mg2+ and the hydrolysis of ATP. X-junction DNA stimulates the ATPase activity o f RecG;, ATPase activity is also stimulated by linear duplex DNA, alth ough to a lesser extent than by X-DNA, but not by linear single-strand ed DNA. In situ 1,10-phenanthroline-copper footprinting shows that Rec G binds to the strand cross-over point at the center of the X-junction . Substrate recognition by RecG was investigated using DNAs that repre sented the various component parts of an X-junction. The minimal DNA s tructure that RecG forms a stable complex with is a flayed duplex, sug gesting that this is the critical feature for junction recognition by RecG. Junction binding and unwinding also depend critically on the con centration of free Mg2+, excess free cation dramatically inhibiting bo th processes. These inhibitory effects are not mediated specifically b y Mg2+; e.g. both Ca2+ and hexamminecobalt(III) chloride also inhibit X-junction binding and unwinding by RecG. The relative abilities of th ese cations to inhibit RecG-junction binding is correlated with their respective abilities to stack X-junction DNA From this we conclude tha t RecG is unable to bind or binds very poorly to fully stacked X-junct ions.