The widely used antitumor drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) reacts with DNA, cross-linking two purine residues through the N7 atoms, which reside in the major groove in B-form DNA. The solution structure of the short duplex [d(CAT-AGCTATG)]2 cross-linked at the GC:GC site was determined by nuclear magnetic resonance (NMR). The deoxyguanosine-bridging cis-diammineplatinum(II) lies in the minor groove, and the complementary deoxycytidines are extrahelical. The double helix is locally reversed to a left-handed form, and the helix is unwound and bent toward the minor groove. These findings were independently confirmed by results from a phase-sensitive gel electrophoresis bending assay. The NMR structure differs markedly from previously proposed models but accounts for the chemical reactivity, the unwinding, and the bending of cis-DDP interstrand cross-linked DNA and may be important in the formation and repair of these cross-links in chromatin.
Using a site-specific, Electron Paramagnetic Resonance (EPR)-active spin probe that is more rigidly locked to the DNA than any previously reported, the internal dynamics of duplex DNAs in solution were studied. EPR spectra of linear duplex DNAs containing 14-100 base pairs were acquired and simulated by the stochastic Liouville equation for anisotropic rotational diffusion using the diffusion tensor for a right circular cylinder. Internal motions have previously been assumed to be on a rapid enough time scale that they caused an averaging of the spin interactions. This assumption, however, was found to be inconsistent with the experimental data. The weakly bending rod model is modified to take into account the finite relaxation times of the internal modes and applied to analyze the EPR spectra. With this modification, the dependence of the oscillation amplitude of the probe on position along the DNA was in good agreement with the predictions of the weakly bending rod theory. From the length and position dependence of the internal flexibility of the DNA, a submicrosecond dynamic bending persistence length of around 1500 to 1700 A was found. Schellman and Harvey (Biophys. Chem. 55:95-114, 1995) have estimated that, out of the total persistence length of duplex DNA, believed to be about 500 A, approximately 1500 A is accounted for by static bends and 750 A by fluctuating bends. A measured dynamic persistence length of around 1500 A leads to the suggestion that there are additional conformations of the DNA that relax on a longer time scale than that accessible by linear CW-EPR. These measurements are the first direct determination of the dynamic flexibility of duplex DNA in 0.1 M salt.
The dG-to-dG, DNA-DNA interstrand cross-link at the duplex sequence 5'-d(GNC) formed by the antitumor drug mechlorethamine (bis(2-chloroethyl)methylamine) was studied both theoretically and experimentally. Computer models of cross-linked DNA were energy minimized using molecular mechanics. The energy minimized structures possessed local distortion of the DNA helix, especially propeller twisting and buckling, caused by the tether length being too small to bridge the spacing of N7 atoms of dG at the sequence 5'-d(GNC) in B DNA. Overwinding of 2-6 degrees was present at each of the two dinucleotide steps spanned by the cross-link. The predicted structural changes were compatible with the possibility that this cross-link would introduce a static bend into the DNA double helix axis. An experimental study provided evidence for this induced bending of the helix axis in interstrand cross-linked samples. DNAs containing multiple mechlorethamine-induced interstrand cross-links exhibited anomalously low electrophoretic mobility in polyacrylamide gels when the lesions were separated by one or two turns. From the degree of gel retardation, the cross-linked DNAs were estimated to be bent by 12.4-16.8 degrees per lesion; estimation of the extent to which this bend was induced by the lesion was complicated by a preexisting bend in the non-cross-linked DNAs used. The data did not allow distinction of a static from an anisotropic dynamic bend; "universal" and "hinge" joints were excluded. Anomalous mobility was maximal when the lesion spacing was 21 bp, suggesting a helical repeat of 10.5 bp per turn.
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