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

Abstract

In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins. Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration. We solved the crystal structure of the Holliday junction bound to a single Escherichia coli RuvA tetramer at 3.1-A resolution. In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center. The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry. Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit. The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding.

Figure 1

Entire view of the RuvA-Holliday junction complex. (A) Top view of the Holliday junction bound to the RuvA tetramer, looking down the protein-DNA interface along the four-fold axis. The DNA molecule is shown in a stick representation colored with the atoms by type: oxygen atoms in red, phosphorus atoms in yellow, nitrogen atoms in blue, and carbon atoms in white. The molecular surface representation shows the RuvA tetramer. (B) Side view of the complex, rotated about 90° from A. The four-fold axis lies perpendicular to the plane at the center of the complex.

Figure 2

Representative protein-Holliday junction interaction. (A) A DNA arm (stick representation) is recognized on the minor groove side by the two HhH motifs (green ribbon representation) of RuvA. The view of the complex is the same as that in Fig. 1B. The junction center is located at the right end of the figure. (B) Close-up view showing the interactions between RuvA and DNA. RuvA is shown in a green-colored stick representation. Hydrogen bonds formed between the protein and the DNA phosphate backbone are indicated by white dotted lines. (C) Schematic representation of protein-DNA interactions. Solid lines indicate polar interactions between the protein and DNA atoms at a distance of less than 3.2 Å. Dotted lines represent candidates for water-mediated interactions within a distance of less than 6.0 Å. (D) Ribbon representations of the single subunit of E. coli RuvA. The subunit of the free form (magenta) is superimposed onto that of the complex with the junction DNA (blue). The blue dot line indicates the structurally disordered connection between the flexible loop and domain III in the complex whereas the connection in the free form structure is not shown. The residues, involved in DNA binding through direct (Lys-84, Gly-117, Lys-119, and Arg-123) or putative indirect (Arg-54 and Leu-113) polar interactions, are indicated on the complex model by their side chains. The side chains of Glu-55 and Asp-56, which form the acidic pin, are also indicated.

Figure 3

2Fo-Fc electron density map (>1.0 σ) showing a DNA duplex, corresponding to one of the four arms of Holliday junction. The DNA molecule is shown in a magenta-colored stick representation. The green wire model indicates the RuvA protein in the complex whereas the yellow ones denote the symmetry-related complex molecules.

Figure 4

Structure of the Holliday junction center. (A) Fo-Fc annealed omit electron density map (>2.5 σ) showing a DNA moiety within the junction center. The two bases closest to the junction center, indicated by a white stick model, were omitted from the map calculation. (B) Environments around unpaired bases in the tetrameric RuvA center. Arg-54, Glu-55, and Asp-56 of each RuvA subunit, which form the acidic pin, are shown by a ball-and-stick representation. (C) Schematic drawing of the Holliday junction structure. The synthetic Holliday junction was designed to form two pairs of opposite arms with different lengths: the north and south arms of a 12-bp DNA duplex and the east and west arms of a 13-bp DNA duplex with a single base overhang at the 5′ end. Two AT base pairs disrupted at the crossover are colored by magenta. The topological features of the unpaired bases may reflect a scene during branch migration, in which the base pair rearrangements are in progress and the new base pairs will be subsequently formed.

Figure 5

Structural comparison between complex I and complex II (16). Octameric complex II is superimposed onto tetrameric complex I, which contains the refined Holliday junction structure. The RuvA tetramer in complex I is represented by the blue ribbon whereas the white ribbons indicate the two tetramers in complex II. Each strand of the junction DNA in complex I is drawn with different ribbon color. The DNA structure in complex II is not shown here because its coordinates are unavailable from the Protein Data Bank.