FUNCTIONAL ANALYSES OF THE DOMAIN-STRUCTURE IN THE HOLLIDAY JUNCTION BINDING-PROTEIN RUVA

Background: Homologous recombination is crucial for genetic: diversity and repairing damaged chromosomes. In Escherichia coli cells, the Ruv A, RuvB and RuvC proteins participate in the processing of an importan t intermediate, the Holliday junction. The RuvA-RuvB protein complex f acilitates branch migration of the junction, depending on ATP hydrolys is. The atomic structure of RuvA should enable critical questions to b e addressed about its specific interactions with the Holliday junction and the RuvB protein, Results: The crystal structure of RuvA shows th e tetrameric molecules with a fourfold axis at the center. Each subuni t consists of three distinct domains, some of which contain important secondary structure elements for DNA binding. Together with the detail ed structural information, the biochemical assays of various mutant Ru vA proteins and domains, isolated by partial proteolysis, allowed us t o define the functional roles of these domains in Holliday junction bi nding and the RuvB interaction, Conclusions: The RuvA molecule is form ed by four identical subunits, each with three domains, I, II and III. The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction, Domain III it; weakly attac hed to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III. Functional analyses of the mutant proteins and the partial digestion products, including H olliday junction binding and branch-migration assays, revealed that do main III and the preceding loop are crucial for RuvB binding and branc h migration, although this region is not required for the junction-DNA binding.