ABSTRACTcis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that binds to and crosslinks DNA. The major DNA adduct of the drug results from coordination of two adjacent guanine bases to platinum to form the intrastrand crosslink cis-[Pt(NH3)2{d(GpG)-N7(1), -N7(2)}] (cis-Pt-GG). In the present study, spectroscopic and calorimetric techniques were employed to characterize the influence of this crosslink on the conformation, thermal stability, and energetics of a site-specifically platinated 20-mer DNA duplex. CD spectroscopic and thermal denaturation data revealed that the crosslink alters the structure of the host duplex, consistent with a shift from a B-like to an A-like conformation; lowers its thermal stability by -9°C; and reduces its thermodynamic stability by 6.3 kcal/mol at 25°C, most of which is enthalpic in origin; but it does not alter the two-state melting behavior exhibited by the parent, unmodified duplex, despite the significant crosslinkinduced changes noted above. The energetic consequences of the cis-Pt-GG crosslink are discussed in relation to the structural perturbations it induces in DNA and to how these crosslinkinduced perturbations might modulate protein binding.The chemotherapeutic efficacy of the anticancer drug cisplatin is derived from its ability to bind and crosslink DNA, the major adduct being the cis-Pt-GG intrastrand crosslink (1). Previous crystallographic, NMR, and gel electrophoretic investigations have evaluated the impact of this crosslink on the structure and conformation of the host duplex (1-9). These studies indicate that formation of the cis-Pt-GG crosslink unwinds DNA by 130and bends it by 34-55°in the direction of the major groove. Avery recent crystallographic study revealed that the crosslink can induce formation of an A-form structural subdomain, thereby creating a hybrid DNA duplex with an A-B junction (8). Such cis-Pt-GG-induced alterations in duplex structure have been implicated in the promotion of specific interactions with cellular proteins that contain one or more high mobility group domainsfor example, HMG1, Ixrl, and human upstream-binding factor (hUBF) (8,(10)(11)(12)(13)(14)(15). When bound by such cellular proteins, the cis-Pt-GG sites are shielded from excision repair (11,13,16,17), thereby enhancing the cytotoxic properties of the drug. Unlike the detailed structural information about the major cisplatin-DNA adduct, comparatively little is known about its corresponding energetic consequences. Such thermodynamic data would reveal how the crosslink influences duplex stability, a property that has been implicated in the modulation of protein recognition and binding (18)(19)(20). To address this deficiency, we have used a combination of calorimetric and spectroscopic techniques to characterize the effect of a single cis-Pt-GG intrastrand crosslink on the conformation, thermal stability, and energetics of a 20-mer DNA duplex. The sequences of unmodified (GG20) and modified (cis-Pt-GG20) duplexes examined are depicted in Fig. 1. Inve...

Proteins containing the high mobility group (HMG) DNA-binding domain form specific complexes with cisplatin-modified DNA which shield the major intrastrand d(GpG) and d(ApG) cross-links from excision repair. The molecular basis for the specificity of binding was investigated for the two isolated domains of HMG1 with a series of 15-bp oligonucleotides, d(CCTCTCN1G*G*N2TCTTC). (GAAGAN3CCN4GAGAGG), where asterisks denote N7-modification of guanosine with cisplatin. Alteration of the nucleotides flanking the platinum lesion modulated HMG1domA recognition in this series by over 2 orders of magnitude and revealed an unprecedented preference for N2 = dA > T > dC. The flanking nucleotide preference for HMG1domB interaction with this oligonucleotide series was less pronounced and had only a 20-fold range of binding affinities. For the N1 = N2 = dA 15-bp probe, 100-fold stronger binding occurred with HMG1domA (Kd = 1.6 +/- 0.2 nM) compared to HMG1domB (Kd = 134 +/- 18 nM). The platinum-dependent recognition of the N1 = N2 = dA 15-bp probe saturates at 1 equiv of HMG1domA and is highly specific, as evidenced by the 1000-fold decrease in HMG1domA binding affinity for the corresponding unplatinated oligonucleotide. HMG domains were unable to bind specifically to cisplatin-modified DNA-RNA hybrids, revealing the need for a deoxyribose sugar backbone for specific complex formation with HMG-domain proteins. Protein-DNA contacts which may account for these observed binding preferences are proposed, and potential implications for the biological processing of cisplatin-DNA adducts are discussed.

Modification of an undecamer deoxyribonucleotide, d(CTCTCGGTCTC), with the paramagnetic cisplatin analogue cis-[Pt(NH3)(4AT)ClI] afforded two orientational isomers of the platinated DNA containing a site-specific intrastrand d(GpG) cross-link. The DNA strand containing the 3‘ orientational isomer was annealed to its complement, and the resulting duplex was investigated by NMR spectroscopy. The structure was determined from conventional NOE studies of the reduced, diamagnetic undecamer and 99 long-range (10−20 Å) electron−proton restraints from the paramagnetic duplex. The platinum-modified duplex is substantially bent toward the major groove. Refinements of the structure with either conventional interproton restraints or a combination of the electron−proton and interproton restraints afforded the same local but different global structures. Both refinements resulted in duplexes that deviated from canonical B-form DNA with widened minor grooves. Addition of the long-range electron−proton restraints allowed for refinement of a duplex structure in excellent agreement with the diamagnetic NMR data (R-factor = 6.08) but exhibiting different positioning of the duplex ends. In particular, the long-range distance restraints afforded a refined duplex with marked similarity (RMSD for all backbone atoms = 1.98 Å) to the tertiary structure of a cisplatin-modified dodecamer duplex solved by X-ray crystallography (J. Am. Chem. Soc. 1996, 118, 12309−21321). The described approach, combining long-range electron−proton and short-range interproton distance restraints in DNA structure refinement, has improved our understanding of the delocalized nature of platinum-induced distortions in duplex DNA and may facilitate high-resolution structural studies of other distorted oligonucleotide duplexes.

The study of the interactions of double-stranded (ds) DNA with the dirhodium carboxylate compounds Rh(2)(O(2)CCH(3))(4)(H(2)O)(2) (Rh1), [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) (Rh2), and Rh(2)(O(2)CCF(3))(4) (Rh3) supports the presence of covalently linked DNA adducts, including stable DNA interstrand cross-links. The present biochemical study refutes earlier claims that no reaction between dirhodium compounds and dsDNA occurs. The reversal behavior of these interstrand cross-links in 5 M urea at 95 degrees C (for different heating times) implies the presence of various coordination modes involving ax/ax, ax/eq, and eq/eq DNA interactions with the dirhodium core. The reaction rates of the dirhodium compounds with dsDNA were determined spectroscopically and are in the order Rh1 << Rh2 < Rh3. This difference in behavior of the three dirhodium compounds correlates with the lability of the leaving groups and corresponds to the extent of interstrand cross-link formation by these compounds on a 123 bp DNA fragment, as observed by denaturing polyacrylamide gel electrophoresis (dPAGE). Since all three dirhodium compounds form covalent Rh-DNA adducts, including interstrand cross-links, it is important that DNA be considered a potential target for biological activity of these dirhodium carboxylate compounds.