Re-pairing DNA: binding of a ruthenium phi complex to a double mismatch

Abstract

We report a crystal structure at atomic resolution (0.9 Å) of a ruthenium complex bound to a consecutive DNA double mismatch, which results in a TA basepair with flipped out thymine, together with the formation of an adenine bulge. The structure shows a form of metalloinsertion interaction of the Λ-[Ru(phen)₂phi]²⁺ (phi = 9,10-phenanthrenediimine) complex at the bulge site. The metal complex interacts with the DNA via the major groove, where specific interactions between the adenines of the DNA and the phen ligands of the complex are formed. One Δ-[Ru(phen)₂phi]²⁺ complex interacts via the minor groove, which shows sandwiching of its phi ligand between the phi ligands of the other two ruthenium complexes, and no interaction of its phen ligands with DNA. To our knowledge, this binding model represents a new form of metalloinsertion in showing major rather than minor groove insertion.

Fig. 1. The TATA-box binding protein, bound to both the TATA-box DNA sequence and an A–C mismatch base pair. Protein shown as grey ribbon. DNA bases use the conventional colour scheme of adenine – red, thymine – blue, guanine – green, cytosine – yellow. The TATA box residues are shown in spacefill mode, and the NAKB colour scheme is used, unless stated, throughout.

Fig. 2. (a) Structural formula of Λ-[Ru(phen)₂phi]²⁺; (b) schematic showing the re-pairing of the bases in the reported structure. The purple blocks highlight the binding sites of the complex. (c) Image showing the large DNA bending. The overall assembly, characterised by a twofold rotational symmetry. Each asymmetric unit is made up of a DNA single strand binding a Λ-[Ru(phen)₂phi]²⁺ with occupancy 1 and a Δ-[Ru(phen)₂phi]²⁺ with occupancy 0.5. The ruthenium complexes are shown in purple.

Fig. 3. The complete assembly (a) the bases of one chain are numbered, with the chain direction indicated. Base T9 is flipped out, and base A5 is ‘bulged’, central base pairs are T4–A8 and T6–A7. The Λ-complex (magenta) binding is stabilised by a hydrogen bond to A7, shown in cyan. The Δ-complex (grey) stacks between the two Λ-complexes, through the phi ligands; (b) and (c) two views showing the stacking of the complexes in the crystal, shown as sticks and semi-transparent spheres, respectively.

Fig. 4. The environment of the Λ-complex. (a) The three adenine bases stacked on the two phenanthroline rings. View towards the phi ligand, which projects into the minor groove, to show the formation of a hydrogen bond (yellow dashes) between one imino H and N3 of A7. The angle formed between the rings of A7 and A8 is 80°; (b) the base pairs T4–A8 and T6–A7, with the stacked ‘bulge’ base A5 and the flipped out base T9, showed in surface mode to highlight the encapsulation of the Λ-complex. The view direction here is into the major groove, with the base G10 also included for clarity; (c) projection onto the phi ligand plane, to suggest how a binding mode similar to this could be possible at an ATA/TAT sequence, if a thymine base were present between A7 and A8.

Fig. 5. The effect of unwinding on the central TA/TA step. (a) The whole assembly with the nucleic acid component shown in surface mode and the metal complexes in spacefill mode; (b) the standard B-DNA model built using the parameters on the w3DNA web server; (c) projection of the TA/TA centre step in this work, showing the extensive pyrimidine-purine ring overlap which results from the unwinding from 36° to 3° (see Table 1); (d) similar projection of the TA/TA centre step in the model duplex, showing the lack of ring overlap; (e) similar projection from step 4 of the Pribnow box structure, PDB code 5EZP.

Fig. 6. Comparison of TA/TA steps. (a) at the surface of the minor groove side of the central TA/TA step (same colour code) showing the contact surface for the Δ-complex in the crystal; (b) the corresponding surface for the model; (c) the corresponding surface (53° twist) in the Pribnow box structure, showing the much smaller cavity; (d) the Δ-complex (spacefill mode) with this surface, showing the hydrophobic interaction with the aromatic edge of the phi ligand of the complex, and only half the surface shown for clarity; (e) details of the interaction geometry shown in stick mode. Colour code used: thymine, blue; adenine, red; cytosine, yellow; and guanine, green.

Fig. 7. Intercalation, insertion, and re-pairing. (a) The mismatched sequence d(CGGAAATTACCG) crystallised with the complex Δ-[Rh(bpy)₂chrysi]³⁺ (PDB code: 2O1I), where chrysi = 5,6-chrysene diimine; (b) the same sequence crystallised with Δ-[Ru(bpy)₂dppz]²⁺; (c) the structure reported here, showing only the Λ-complex for clarity and ease of comparison.