Crystal structure of E.coli RuvA with bound DNA Holliday junction at 6 Å resolution

Abstract

Here we present the crystal structure of the Escherichia coli protein RuvA bound to a key DNA intermediate in recombination, the Holliday junction. The structure, solved by isomorphous replacement and density modification at 6 A resolution, reveals the molecular architecture at the heart of the branch migration and resolution reactions required to process Holliday intermediates into recombinant DNA molecules. It also reveals directly for the first time the structure of the Holliday junction. A single RuvA tetramer is bound to one face of a junction whose four DNA duplex arms are arranged in an open and essentially four-fold symmetric conformation. Protein-DNA contacts are mediated by two copies of a helix-hairpin-helix motif per RuvA subunit that contact the phosphate backbone in a very similar manner. The open structure of the junction stabilized by RuvA binding exposes a DNA surface that could be bound by the RuvC endonuclease to promote resolution.

Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer

“…1 A and B), as previously revealed by the 6-Å resolution structural analysis of a different complex with shorter DNA arms (15). The junction DNA conforms well to the positively charged concave surface of the RuvA tetramer.…”

“…One RuvA subunit covers eight base pairs of a single DNA arm starting at the crossover point. The junction DNA bound to RuvA adopts a remarkable conformation, which substantially deviates from the strict square-planar arrangement, as observed in the previous lower resolution structure (15). The Holliday junction is most depressed in close vicinity of the crossover point, to allow intimate contact between the DNA interface and the RuvA tetramer.…”

“…4 A and C), although the previous structural analysis at 6-Å resolution suggested that the base pairs in the corresponding region were maintained (15). The configurations of these unpaired bases seem to hint at a possible intermediate DNA structure during branch migration, where two base pairs, located on opposite sides across the junction center, are disrupted and subsequently reformed with other partners in the adjacent arms (Fig.…”

“…They both contain a single junction DNA but different numbers of the RuvA tetramer, one tetramer for complex I and two tetramers for complex II. The crystal structure of the E. coli RuvA-Holliday junction complex in the complex I form was solved at 6-Å resolution, and an overall structural view of the complex was reported (15). More recently, the crystal structure of octameric complex II from Mycobacterium leprae has been determined at 3.0-Å resolution (16).…”

Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

“…Genetic studies showed that RuvC cannot function in vivo in the absence of RuvAB (7,8), and it has been proposed that an RuvABC complex, known as the resolvasome, allows RuvC to scan for cleavable sequences (3, 9 -11). RuvA binds to HJs in vitro as one tetramer (complex I) or two tetramers that sandwich the junction (complex II) in a concentration-dependent manner; however, it is not clear whether the RuvAB complex contains one or two tetramers of RuvA in vivo (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). A RuvAB branch migration complex made of two RuvA tetramers would prevent access of RuvC to the Holliday junction.…”