The acidic pin of RuvA modulates Holliday junction binding and processing by the RuvABC resolvasome

Holliday junctions are four-way branched DNA structures formed during recom bination, replication and repair. They are processed in Escherichia coli by the RuvA, RuvB and RuvC proteins. RuvA targets the junction and facilitate s loading of RuvB helicase and RuvC endonuclease to form complexes that cat alyse junction branch migration (RuvAB) and resolution (RuvABC). We investi gated the role of RuvA in these reactions and in particular the part played by the acidic pin located on its DNA-binding surface. By making appropriat e substitutions of two key amino acids (Glu55 and Asp56), we altered the ch arge on the pin and investigated how this affected junction binding and pro cessing. We show that two negative charges on each subunit of the pin are c rucial. They facilitate junction targeting by preventing binding to duplex DNA and also constrain branch migration by RuvAB in a manner critical for j unction processing. These findings provide the first direct evidence that R uvA has a mechanistic role in branch migration. They also provide insight i nto the coupling of branch migration and resolution by the RuvABC resolvaso me.