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. 2001 Dec 3;20(23):6601-11.
doi: 10.1093/emboj/20.23.6601.

Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2

S Ceschini  1 , A Keeley, M S McAlister, M Oram, J Phelan, L H Pearl, I R Tsaneva, T E Barrett
Affiliations

Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2

S Ceschini et al. EMBO J. .

Abstract

Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.

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Figures

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Fig. 1. Ydc2 structure. (A) Secondary structure cartoon of the Ydc2 monomer. This and other molecular graphics figures were generated using Bobscript (Robert Esnouf’s adaptation of Molscript; Kraulis, 1991) and rendered using RASTER3D (Merrit and Murphy, 1994), except for Figures 3A and 7B, which were generated using GRASP (Nicholls et al., 1993). (B) Topology of the secondary structural elements. (C) Secondary structure cartoon of the Ydc2 dimer observed in the crystals, viewed down the non-crystallographic 2-fold axis. The significant surface area burial and conservation of the interface suggest that this is the biologically authentic dimer.
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Fig. 2. Structural homology of Ydc2 and RuvC. (A) Secondary structure cartoons of Ydc2 (left) and E.coli RuvC (right) colour-ramped blue→red from the N- to C-termini. The structures were superimposed using SSAP (Orengo et al., 1992) and separated for clarity. (B) Structure-based sequence alignment of Ydc2 and E.coli RuvC. Residues implicated in catalysis and conserved between Ydc2 and RuvC are highlighted in magenta.
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Fig. 3. DNA-binding surface and active site of Ydc2. (A) Orthogonal views of the molecular surface of the Ydc2 dimer coloured (blue→red, +ve→–ve) according to electrostatic potential. One face (left) shows a substantial excess of basic residues consistent with DNA binding, whilst the recessed negative patches are formed by a cluster of conserved acidic residues (red arrows). The ‘pin’ segments connecting strand d and helix 4 protrude from this face (black arrows). The opposite face (right) has a more acidic surface. (B) Conserved residues forming the acidic patch from Ydc2 (yellow) structurally aligned with the topologically equivalent residues from RuvC (green). Mutagenesis and crystallographic studies of RuvC implicate Asp7 and Asp141 as essential ligands for a catalytic metal ion.
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Fig. 4. Identification and mutagenesis of catalytic residues. (A) Bound metal ion observed in one Ydc2 monomer. The coordination is most consistent with a calcium ion, but the site would probably accommodate two Mg2+ ions during catalysis. The electron density shown is from an Fo – Fc map contoured at 2.5σ. (B) Mutation of either of the direct metal ligands Asp46 or Asp230 to asparagine completely abolishes the ability of Ydc2 to resolve Holliday junctions in the presence of Mg2+, compared with the wild-type enzyme, which efficiently releases nicked duplexes under the same reaction conditions. (C) Both mutants and the wild-type enzyme are able to bind to Holliday junctions in the absence of Mg2+, implying purely catalytic roles for Asp46 and Asp230.
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Fig. 5. Active site variability and communication. (A) Superposition of the active sites from the metal-ion bound monomer (yellow) and the metal-free monomer (green). Significant changes in conformation of side chains and in the order of adjacent segments of the polypeptide chain occur as a result of metal ion binding. (B) A pathway for communication between the two active sites is provided by the direct interaction of the N-termini of helix 4 at the dimer interface. These are directly linked to the flexible ‘pin’ segments, which in turn connect to the active site metal ion ligand Glu117. Changes in the conformation of one active site would be communicated to the other site via this pathway, and could mediate the positive cooperativity observed between the first and second strand cleavage reactions.
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Fig. 6. Holliday junction complex. Model of a productive Ydc2–Holliday junction complex. A model Holliday junction in an open, approximately square-planar conformation (transparent CPK model) can be docked onto the basic face of the Ydc2 dimer, bringing the scissile phosphodiester bonds close to the acidic cluster and the metal ion-binding site (red). The ‘pin’ and N-terminus of helix 4 that protrude from this face are accommodated by the centre of the open junction, but would prevent binding of the junction in a stacked-X conformation. Scissile phosphate groups are highlighted in yellow.
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Fig. 7. Convergent ‘S’-shaped structures for Ydc2 and T4 endo VII. Ydc2 (left) and bacteriophage T4 endo VII (right) show a remarkable similarity in their overall shape, despite totally unrelated architectures and folds. Both have an ‘S’ shape when viewed towards their basic DNA-binding surfaces, and both have basic triple-helix domains extending out from their core. However, they have very different specificity for the conformation of their Holliday junction substrate.
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