Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions

Branch migration of Holliday junctions, which are central DNA intermediates in homologous recombination, is promoted by the RuvA-RuvB protein complex, and the junctions are resolved by the action of the RuvC protein in Escher ichia coli. We report here the cloning of the ruvB gene from a thermophilic eubacterium, Thermus thermophilus HB8 (Tth), and the biochemical character ization of the gene product expressed in E. coli. The Tth ruvB gene could n ot complement the UV sensitivity of an E. coli ruvB deletion mutant and mad e the wild-type strain more sensitive to UV. In contrast to E. coli RuvB, w hose ATPase activity is strongly enhanced by supercoiled DNA but only weakl y enhanced by linear duplex DNA, the ATPase activity of Tth RuvB was effici ently and equally enhanced by supercoiled and linear duplex DNA. Tth RuvB h ydrolyzed a broader range of nucleoside triphosphates than E. coli RuvB. In addition, Tth RuvB, in the absence of RuvA protein, promoted branch migrat ion of a synthetic Holliday junction at 60 degrees C in an ATP-dependent ma nner. The protein, as judged by its ATPase activity, required ATP for therm ostability. Since a RuvA protein has not yet been identified in T. thermoph ilus, we used E. coli RuvA to examine the effects of RuvA on the activities of Tth RuvB. E: coli RuvA greatly enhanced the ability of Tth RuvB to hydr olyze ATP in the presence of DNA and to promote branch migration of a synth etic Holliday junction at 37 degrees C. These results indicate the conserva tion of the RuvA-RuvB interaction in different bacterial species, and sugge st the existence of a ruvA homolog in T. thermophilus. Although GTP and dGT P were efficiently hydrolyzed by Trh RuvB, these nucleoside triphosphates c ould not be utilized for branch migration-in vitro, implying that the confo rmational change in RuvB brought about by ATP hydrolysis, which is necessar y for driving the Holliday junction branch migration, cannot be accomplishe d by the hydrolysis of these nucleoside triphosphates.