Staggered intercalation of DNA duplexes with base-pair modulation by two distinct drug molecules induces asymmetric backbone twisting and structure polymorphism

Abstract

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson-Crick base pair at the two drug-binding interfaces can result in a single-stranded 'chair-shaped' DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a 'curved-shaped' duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs' potential synergetic use.

Figure 1.

Chemical structures of (A) actinomycin D (ActD) and (B) echinomycin (Echi). The chromophore phenoxazone (PXZ) and two cyclic peptides [α] and [β] in the structure of ActD and the two quinoxaline moieties (QUI) of Echi are shown. The other abbreviations shown in the figure represent l-threonine (THR), d-valine (DVA), l-proline (PRO), sarcosine (SAR), N-methyl-l-valine (MVA), d-serine (DSN), l-alanine (ALA), N-dimethyl-l-cysteine (N2C) and N-methyl-l-cysteine (NCY). The numbers indicate the order of the cyclic peptides in ActD and Echi structures. (C) Schematic representation of ActD-Echi complexed with various mismatch-containing duplexes. Base numbering in chain A and complementary chain B is maintained throughout the study. Central X4:X11 (highlighted in bold red) represents mismatches or Watson–Crick base pair that occurred in this study. PXZ (dark blue) represents the phenoxazone ring of ActD intercalated between the G2pC3/G12pC13 steps; QUI0 and QUI1 (green) are the two quinoxaline rings of Echi intercalated between the C5pG6/C9pG10 steps.

Figure 2.

(A) Biological assembly of a crystal structure with a central A:G mismatch in a DNA duplex complexed with ActD and Echi, as shown in front view (left) and side view (right), exhibiting orthogonal asymmetric single-stranded backbone distortion. The DNA backbone and bases are coloured grey. Bases A4 and G11 form a mismatch and are coloured orange and cyan. An enlarged view of the 2F_o– F_c electron density map of the A:G mismatch is shown on the right. (B) The overall crystal structures of two G:A mismatch containing complexes, G:A-CPX1 and G:A-CPX2 with ActD and Echi are shown in pink and orange cartoon representations, respectively. Bases G4 and A11 are highlighted with cyan and orange colours. Superimposition of DNA duplexes in these complexes shows significant differences in the backbone shape with an average r.m.s.d. of 2 Å between the two complexes. An enlarged view of the 2F_o– F_c electron density map of the two distinct G:A mismatches are shown at the bottom.

Figure 3.

(A) Overall backbone comparison between A:G and G:A mismatch complexes. The A:G complex (grey cartoon) and G:A-CPX1 (pink cartoon) show twisting of only chain B, with the helical axes remaining straight in Chain A. This situation form a ‘chair’ shaped backbone conformation. However, the DNA backbone shows a sharp bend, resulting in a ‘curved’ duplex upon intercalation of ActD and Echi in G:A-CPX2 (orange cartoon). (B) Comparison of central mismatch geometries in A:G and G:A mismatch complexes. The A4:G11 mismatch adopts a common anti–anti geometry with two hydrogen bonds, while the G4:A11 pair in G:A-CPX1 forms a typical syn–anti type wobble base pair with a single hydrogen bond. G4:A11 mismatched pair in G:A-CPX2 forms a sugar-edged ‘syn-like’ anti-anti geometry with a single hydrogen bond. Hydrogen bonds are shown as black dotted lines, and numbers indicate distances in angstroms (Å). Differences in base pair geometries also lead to different C1'–C1' distances at mismatch sites.

Figure 4.

Overall stacking and hydrogen bonding interactions between ActD (dark blue sticks) and Echi (green sticks) and DNA nucleotides in the A:G (light blue sticks), G:A-CPX1 (pink sticks) and G:A-CPX2 (orange sticks) mismatched complex structures. (A) The phenoxazone ring (PXZ) of ActD is intercalated into the G2pC3 step, forming stacking and hydrogen bonding with the bases G2, C13 and C3 in the three complexes. (B) Intermolecular hydrogen bonds to the N2/N3 atoms of bases G2 and G12 and the threonine residue (THR of ActD) in all three complexes are shown. (C) The quinoxaline ring (QUI) and alanine residue (ALA) of Echi are intercalated into the C5pG6 sites, resulting in stacking and hydrogen bonding.

Figure 5.

Overall structure of C:G and G:C Watson–Crick duplexes in complexes with ActD and Echi. (A) Biological assembly of the crystal structure containing a central C:G Watson–Crick base pair in a DNA duplex complexed with ActD and Echi, as shown in front (left) and side (right) views, with typical single-stranded backbone distortion. The DNA backbone is shown in a white cartoon with the central C4 and G11 bases highlighted in bold red and cyan font. (B) Biological assembly of the crystal structure containing a G:C Watson–Crick base pair shows two separate duplexes after ActD and Echi intercalation, as shown in the orange cartoon. G:C-CPX1 displays backbone with twisted end, while G:C-CPX2 shows a kink in individual DNA backbone strand. The central bases G4 and C11 are highlighted in cyan and red bold. (C) Comparison between the geometries of the central C4:G11 and G4:C11 pairs in the two complex structures. The C4:G11 base pair shows a typical Watson–Crick geometry with both bases in the anti-anti conformations with three hydrogen bonds. The G4:C11 base pair, on the other hand, forms a Hoogsteen base pairing with syn-anti geometries containing two different χ values for syn G4 bases and hydrogen-bonded interactions. The differences in the C1'-C1' distances for each base pair type are shown. Hydrogen bonds are represented by black dotted lines and distances in angstroms (Å).

Figure 6.

(A) Overall biological assembly of a discontinuous four-way junction superstructure formed by two independent duplexes of the G:C base pair complex structure through symmetrically related duplexes. The outer strands in the four-way junction structure are coloured in cyan, while the inner chains forming a crossover junction are shown in pink cartoons. The ActD and Echi are shown in blue and green sticks. Asterisk (*) symbols indicate symmetry-related complexes or drug molecules. (B) Schematic diagram of the superstructure of the four-way junction with detailed numbering of residues and insertion sites for ActD (blue) and Echi (green). (C) The core of the superstructure formed by the intersection of two symmetry-related complexes (CPX1 and CPX1*), viewed from above and from the front. A side view of the crossover junction shows that the two ActD molecules are arranged at an angle of about 62° to the plane of the phenoxazone (PXZ) rings. (D) Detailed hydrogen bonding (black dotted lines) and stacking interactions (green dashed lines) of PXZ and THR residues of ActD with G12pC13/G2*pC3 residues stabilize the junction site.

Figure 7.

Comparison of DNA topologies of four-way junction superstructures in different sequences induced by intercalators. (A) The ‘X’-shaped crossover topology generated in a pseudo four-way junction superstructure induced by a platinum-based [Pt(H₂bapbpy)]-(PF₆)₂ compound. (B) When a triaminotriazine DNA intercalator is inserted into the T:T mismatch duplex, a ‘U’-shaped head-to-head four-way junction like topology is formed. (C) Psoralen-based 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) DNA intercalation compound induces a Holliday junction by cross-linking with thymine bases to form an antiparallel stacked topology. (D) Intercalation of ActD induces a discontinuous four-way junction with an antiparallel alignment of the DNA backbone strands. Cyclic peptide parts of ActD and Echi has been removed to enhanced clarity.