In vitro reconstitution of the late steps of genetic recombination in E. coli.

Abstract

Purified proteins have been used to reconstitute an in vitro system for the medial-to-late stages of recombination in E. coli. In this system, RecA protein formed recombination intermediates that were processed by the actions of the RuvA, RuvB, and RuvC proteins. RuvAB was found to promote branch migration, to dissociate the RecA filament, and to modulate the orientation of cleavage of Holliday junction resolution by RuvC. Monoclonal antibodies directed against RuvA, RuvB, or RuvC inhibited resolution in the reconstituted system. Specific protein-protein interactions between the branch migration motor (RuvB) and the resolvase (RuvC) were also observed. These results provide evidence for coordinated action during the late stages of recombination, possibly involving the assembly of a RuvABC branch migration/resolution complex.