Chacterization of the ternary complexes formed in the reacton of cis-diamminedichloro-platinum ( II ) , ethidium bromide and nucleic acids

Content Provider	Semantic Scholar
Author	Malinge, Jean-Marc Schwartz, Annie
Abstract	The purpose of this study was to characterize the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II) (cis-DDP) and nucleic acids, in the presence of the intercalating compound ethidium bromide (EtBr). In these ternary complexes, some EtBr is tightly bound to the nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or thin layer chromatography at basic pH. These ternary complexes are formed with double stranded but not with single stranded nucleic acids. They are not formed if cis-DDP is replaced by transdiamminedichloroplatinum(II). The amount of tightly bound EtBr depends upon the sequence of the nucleic acid, being larger with poly (dG-dC).poly(dG-dC) than with poly(dG).poly(dC). Spectroscopic results support the hypothesis that the tight binding of the dye is due to the formation of a bidentate adduct (guanine-EtBr)cis-platin. The visible spectrum of the ternary complexes is blue-shifted as compared to that of EtBr intercalated between the base pairs of unplatinated DNA and it depends upon the conformation of the ternary complex. The fluorescence quantum yield of the ternary complexes is lower than that of free EtBr in water. Tightly bound EtBr stabilizes strongly the B form versus the Z form of the ternary complex poly(dG-dC)-Pt-EtBr and slows down the transition from the B form towards the Z form. The sequence specificity of cis-DDP binding to a DNA restriction fragment in the absence or presence of EtBr is mapped by means of the 3's 5' exonuclease activity of T4 DNA polymerase. In the absence of the dye, all the d(GpG) sites and all the d(ApG) sites but one in the sequence d(TpGpApGpC) are platinated. The d(GpA) sites are not platinated. In the presence of EtBr, some new sites are detected. These results might help to explain the synergism for drugs used in combination with cis-DDP and in the design of new chemotherapeutic agents. INTRODUCTION Numerous studies have been devoted to the study of the reaction of the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA. The mechanism of cis-DDP action is not yet known but it is often thought that its antitumor activity is related to its binding to DNA (1-4) . In the in vitro reaction between cis-DDP and DNA, the two major adducts derive from intrastrand cross-links of cis-DDP on d(GpG) and d(ApG). Minor adducts derive from intrastrand cross-links of cis-DDP on two guanine residues separated by one C I RL Press Limited, Oxford, England. 1 779 Nucleic Acids Research base and from interstrand cross-links of cis-DDP on two guanine residues (512). The DNA conformation plays an important role in the binding of the drug. It has be found that the relative percentages of adducts depend on whether cis-DDP reacts with native or denatured DNA (12). In the reaction of cis-DDP and poly(dG-m5dC).poly(dG-m5dC) a bidentate or a monodentate adduct is formed depending on whether the polynucleotide is in the B or in the Z form (13). The presence of intercalating dyes can interfere with both the position and the mode of cis-DDP binding to DNA. By means of exonuclease III mapping experiments, it has been proposed that intercalating dyes such as ethidium bromide can modulate the local structure of DNA and thus influence the selectivity of cis-DDP binding (14-16). In a recent work we have found that incubation of double stranded nucleic acids and cis-DDP in the presence of some intercalating dyes such as ethidium bromide or proflavine leads to the formation of ternary complexes in which the dyes are tightly bound to the nucleic acids (17). Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or by thin layer chromatography at basic pH. To explain this strong binding, it has been suggested that cisDDP cross-links a guanine residue and a dye. Moreover, it has been shown by competition experiments that ethidium bromide, proflavine and also acridine (which does not form a tightly bound complex) interfere with the preferential binding of cis-DDP to (dG)n.(dC)n sequences. We here report some more results on the tightly bound complexes formed between natural and synthetic double stranded polynucleotides, cis-DDP and ethidium bromide. Spectroscopic studies (visible absorption, fluorescence and circular dichroism) show that the dye and platinum are in close contact. The tightly bound dye stabilizes the B form versus the Z form of the ternary complex poly(dG-dC)-Pt-EtBr and reduces the transition rate from the B form to the Z form. The sequence specificity of platinum binding in the absence and in the presence of ethidium bromide was mapped by means of the 3' + 5' exonuclease activity of T4 DNA polymerase. MATERIALS AND METHODS Double stranded polynucleotides, purchased from Boehringer Manheim and Pharmacia, Micrococcus luteus DNA prepared as already described (18) were treated twice with phenol and then precipitated with ethanol. Stock solutions of nucleic acids were made in 10 mM NaCl04/1 mM phosphate buffer pH 7.5. Unlabelled ethidium bromide (Sigma) and [C-14] ethidium bromide (0.7
File Format	PDF HTM / HTML
Alternate Webpage(s)	http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC340581&blobtype=pdf
Language	English
Access Restriction	Open
Content Type	Text
Resource Type	Article