title: Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution.
abstract: Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the 'nick-counter-nick' mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.
authors.affiliation: Department of Biochemistry, Molecular biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Luan
authors.initials: L
authors.lastname: Chen
authors.forename: Ke
authors.initials: K
authors.lastname: Shi
authors.forename: Zhiqi
authors.initials: Z
authors.lastname: Yin
authors.forename: Hideki
authors.initials: H
authors.lastname: Aihara
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D045566:DNA, Cruciform / Q000378:metabolism*; D019281:Dimerization; D045565:Holliday Junction Resolvases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D045566:DNA, Cruciform; D004247:DNA; D045565:Holliday Junction Resolvases