Construction, Purification and Characterization of Novel, Fluorescent RuvA Chimeras

The Escherichia coli RuvA and RuvB proteins play important roles in the late stages of recombinational DNA repair and genetic recombination. RuvB is a DNA-stimulated helicase, whose activity is controlled by the homotetrameric RuvA protein. In order to facilitate the studies of the interaction and the role of these proteins both in vivo and in vitro, we constructed a series of novel, RuvAautofluorescent protein fusions. The fusions were then expressed with a “wild type” RuvA using a dual plasmid expression system. The resulting heterogeneous populations of chimeras are readily separated using chromatography. The purified chimeras contain one to four fluorescent tagged subunits and maintain full functionality with RuvB in the presence of Holliday junction substrates.