Polymorphic G:G mismatches act as hotspots for inducing right-handed Z DNA by DNA intercalation

Abstract

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.

Figure 1.

Overall structure of the Co^II-(Chro)₂–d(TTGGCGAA)₂ complex. (A) Chemical structure of the Co^II-(Chro)₂ dimer complex. (B) Refined structure of the Co^II-(Chro)₂–d(TTGGCGAA)₂ complex viewed from the major groove. The asymmetric unit contains three independent complexes (CPX1, CPX2, and CPX3). The DNA duplex is represented in the purple cartoon, cobalt(II) ions are shown as pink spheres, and (Chro)₂ are shown in brown and sky-blue sticks. Thymines are coloured in yellow, guanines in red, cytosines in blue, and adenines in green. (C) The d(TTGGCGAA)₂ duplex backbone is represented as red and blue sticks in all three complexes (CPX1, CPX2, and CPX3). (D) Superimposition of the overall structure between CPX1 (green), CPX2 (red), and CPX3 (blue) of d(TTGGCGAA)₂ duplexes with modelled B-form DNA d(TTGGCGAA/TTCGCCAA) (yellow). (E) Superimposition of the [Co^II(Chro)₂] ligand from three independent complexes: CPX1 (green), CPX2 (red) and CPX3 (blue).

Figure 2.

Geometry of G:G mismatches in Co^II-(Chro)₂–DNA complex. (A–C) Cartoon representations of all three independent DNA duplexes present in an asymmetric unit of the Co^II-(Chro)₂–DNA complex. The DNA backbone is represented as silver arrows, adenine in green, thymine in orange, cytosine in red and guanine in blue. The refined (2F_o-F_c) difference Fourier electron density map shows G:G mismatched pairs of the Co^II-(Chro)₂–d(TTGGCGAA)₂ complex, contoured at 1.0 σ. Hydrogen bonds are represented by dotted lines, with numbers indicating the distance between the two contributing atoms in Angstroms (Å).

Figure 3.

DNA rearrangement and right-handed Z DNA formation in the G:G mismatched DNA duplex forced by actinomycin D. (A) Chemical structure of actinomycin D (ActiD). (B) Biological assembly and schematic representation of the ActiD–d(TTGGCGAA)₂ complex viewed from the front and a side-view. The bases are numbered from T1 to A8; the bases in the asymmetric unit are numbered with an asterisk (*) sign. The DNA backbone is coloured blue, adenine in green, thymine in yellow, cytosine in blue and guanine in red. (C) Refined (2F_o-F_c) difference Fourier electron density map showing A:G and G:A mismatched base pairs of the ActiD–d(TTGGCGAA)₂ complex. (D) Comparison of the central core of the d(TTGGCGAA)₂ DNA backbone with the d(CGCGCG)₂ Z-DNA structure (PDB ID: 2DCG). (E) Twist and Roll angle parameter comparison for DNA backbones of the ActiD–d(TTGGCGAA)₂ complex and the d(CGCGCG)₂ Z-DNA. Values for perfect A- and B-DNA helices are also shown for comparison.

Figure 4.

Skeletal models showing the antibiotic binding sites in the actinomycin D (ActiD)–DNA complex. Close-up view of the ActiD-TTGGC part (ActiD shown as ball-and-stick and DNA as skeletal representation). The two phenoxazone rings (PXZ) are intercalated individually into the (G3pC5)-(G6*pA7*) step in (A) and the (G3*pC5*)-(G6pA7) step in (B), respectively. (C–E) Detailed conformation showing the stacking interactions in the ActiD–DNA complex at various base-pair steps of the refined structure. Hydrogen bonding is marked by black dotted lines. The phenoxazone ring of ActiD (yellow) and ActiD* (green) is intercalated at the A7:G3*/G6:C5* base pairs from the minor groove side by pushing out G4 (and G4*) in (C). The stacking interactions between the phenoxazone ring and C5*:G6/ A7:G3* base pairs is shown in (D). The flipped-out G4 form a single hydrogen bond between G4:C5 and G4*:C5*. The intermolecular hydrogen bonds between G3-N3 and the NH of Thr7 and G6-N3 Thr1 of the two ActiD moieties are shown in (E).

Figure 5.

Water-cluster, triplet-stranded base pair formation and Na⁺-mediated hydrogen bonding stabilizing the ActiD–DNA complex structure. (A) Water-mediated hydrogen bonding of the flipped out guanines, stabilizing the overall structure of the ActiD–DNA complex. (B) Coordination of the metal ions in the ActiD–DNA complex. The 2F_o-F_c electron density is contoured at 1.0 σ. The coordinated metal ions and water molecules appear clearly in the refined structures between the symmetry units. Coordination and hydrogen bonds are shown by dashed lines. The hydrated Na⁺ ion, with ∼50% occupancy in the complex, coordinates with two symmetry-related actinomycin residues, having square-planar antiprismatic geometry. The terminal bases form a (T:A):T triplet with the symmetry-related strands. In a triplet, adenine forming a Watson–Crick base pair and a Hoogsteen base pair with thymine.

Figure 6.

Close-up views of G3:G14 mismatched pairs in the Co^II-(Chro)₂–d(TTGGCGAA)₂ structure showing the base-pair geometry, stacking interactions and hydrogen bonding patterns. (A) The G3(syn):G14(syn) pair in CPX1 is stabilized by amide–Π stacking interactions (shown in red-dashed lines) between N1 of the G3 and G4 ring and the van der Waals interactions between the oxygen of the C6 carbonyl group of the G3 and G14 bases (indicated by green-dashed lines). (B) Water-mediated hydrogen bonds between the N2 atom of G3(anti) and N7 of G14(syn) bases are represented as black dotted lines in CPX2, where the water is shown as a cyan sphere. (C) Distorted geometry between the G3(anti):G14(syn) shows that the syn–syn-like characteristic is stabilized by a single hydrogen bond between the N1 of G3 and the oxygen of the C6 carbonyl group in CPX3.

Figure 7.

Comparison of DNA bending in different sequence context associated with ligand binding. The marked kink induced by (A). ActiD in the d(TTGGCGAA)₂ complex (B). λ-[Ru-(TAP)₂(dppz)]²⁺ in the d(TCGGCGCCGA)₂ complex (C). λ–[Ru(phen)₂(dppz)]²⁺ in the d(CCGGTACCGG)₂ complex and (D) echinomycin in the d(ACGTCGT)₂ complex, at the single-step in various DNA duplexes. (E) The sharp bend associated with the ActiD ligand bound to the neighbouring GpC sites flanking a G:G mismatch in the d(ATGCGGCAT)₂ duplex is due to the sum of roll angles of three different steps at the intercalation site. The DNA bending angles are measured in terms of the respective roll angles (°) at the kinked step. The lower figure shows a schematic representation of ligand-induced DNA bending due to different intercalators in each duplex. The DNA bases are coloured as adenine-green, thymine-pink, cytosine-yellow, and guanine-blue. The respective PDB IDs for each structure are given in bold, red letters.