Drug-induced DNA repair: X-ray structure of a DNA-ditercalinium complex.

Abstract

Ditercalinium is a synthetic anticancer drug that binds to DNA by bis-intercalation and activates DNA repair processes. In prokaryotes, noncovalent DNAditercalinium complexes are incorrectly recognized by the uvrABC repair system as covalent lesions on DNA. In eukaryotes, mitochondrial DNA is degraded by excess and futile DNA repair. Using x-ray crystallography, we have determined, to 1.7 A resolution, the three-dimensional structure of a complex of ditercalinium bound to the double-stranded DNA fragment [d(CGCG)J2. The DNA in the complex with ditercalinium is kinked (by 15') and severely unwound (by 36) with exceptionally wide major and minor grooves. Recognition of the DNA-ditercalinium complex by uvrABC in prokaryotes, and by mitochondrial DNA repair systems in eukaryotes, might be related to drug-induced distortion of the DNA helix. and length (7) of the linker. Furthermore, the drug is inactivated when the N-7 position, or to a lesser extent the C-6 position, is substituted with alkyl groups larger than methyl (8), or when the position of the nitrogen at the 7 position within the chromophore is altered (5).To understand the mechanisms ofDNA repair induction by ditercalinium and why minor modifications of the drug result in dramatic changes in activity, we need to establish the details of how the drug interacts with and distorts DNA. Using x-ray crystallography, we have determined, to 1.7 A resolution, the three-dimensional structure of a complex of ditercalinium bound to the double-stranded DNA fragment [d(CGCG)h2. § A series of NMR studies of DNA-ditercalinium complexes by Roques and coworkers (9-13) has allowed us to compare those results with the x-ray structure described here. The three-dimensional structures of these DNA-drug complexes provide us with the potential to better understand the molecular basis offunction and recognition in DNA repair processes. MATERIALS AND METHODSThe self-complementary DNA tetramer d(CGCG) was synthesized by the phosphotriester method and purified with Sep-Pak C18 cartridges (Waters). Ditercalinium was kindly supplied by Bernard P. Roques (Paris). Crystals were grown at room temperature in sitting drops by the vapor diffusion method. The crystallization mother liquor initially contained 0.7 mM DNA (single-strand concentration), 16.8 mM sodium cacodylate buffer (pH 6.0), 14 mM ammonium acetate, 0.3 mM spermine tetrahydrochloride, 0.8 mM magnesium chloride, 6% 2-methyl-2,4-pentanediol, and 0.2 mM ditercalinium. The sitting drops were equilibrated against a reservoir of 30% 2-methyl-2,4-pentanediol. Yellow 2422The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Molecular Dynamics Simulations of the Bis-Intercalated Complexes of Ditercalinium and Flexi-Di with the Hexanucleotide d(GCGCGC)2: Theoretical Analysis of the Interaction and Rationale for the Sequence Binding Specificity

“…This discrimination can account for the experimentally observed poor van der Waals contacts of the chromophores with the internal cytosines. [21][22][23] With regard to the electrostatic term of the stacking interaction between the chromophores and the bases that delimit the bis-intercalation sites, they are consistently repulsive in both crystal structures, which could account for the absence of van der Waals contacts with the external cytosines and for the observed bending of the helix.…”

“…We chose to represent a In order to add a terminal G:C base pair on both 5′-and 3′-ends, a symmetry related copy was generated on each side of the helix by applying the appropriate symmetry operators as the complexes are aligned with the 5′-ends of a given strand stacked over the 3′-end of the adjacent strand so that infinite helices are formed in the crystal lattice. [21][22][23] The advantages of this procedure are that the stacking interactions are already optimized and that it is relatively easy to add the extra bridging phosphate in a suitable geometry. A short optimization run restraining the ligand and base atoms to their initial coordinates allowed readjustment of the sugar-phosphate backbone while maintaining the overall conformation of the complexes.…”

“…34,35 In the case of the ditercalinium complex, initial difficulties for solving the structure by molecular replacement were precisely due to a wrong positioning of the linker, and the crystal was later found to diffract to only 2.5 Å resolution in the cell direction approximately perpendicular to the DNA helical axis. 21 The Flexi-Di linker is reported to have been constructed from coordinates of a spermine molecule and added to the model; 23 this fact, together with the intermediate resolution of this crystal structure, precludes the accurate location of the linker atoms, which nevertheless are highly disordered. We thus felt it was justified to reoptimize the geometry of both linkers.…”

“…The X-ray crystal structures of the complexes between d(CGCG)2 and ditercalinium (1.7 Å resolution, R-factor ) 22.5%), 21 and between d( Br CGCG)2 and Flexi-Di (2.5 Å resolution, R-factor ) 23.9%) 23 were retrieved from the Brookhaven Data Bank. 27 Hydrogen atoms were added to the molecules using standard geometries.…”

“…21,22 Among the most remarkable features found are (1) complete lack of van der Waals contacts between the bases of the terminal cytidines and the chromophores of ditercalinium; (2) poor stacking of the drug's chromophore on the internal cytosines; (3) lack of 2-fold symmetry in the conformation and interactions of the two halves of the complex; (4) lack of van der Waals contacts between the cytosines and the bis(ethylpiperidinium) linker, which can form only one hydrogen bond with the DNA major groove; and (5) significant bending of the DNA helical axis toward the minor groove (∼15°kink).…”