Chemical probes of the conformation of DNA modified by cis-diamminedichloroplatinum(II)

Abstract

The purpose of this work was to analyze at the nucleotide level the distortions induced by the binding of cis-diamminedichloroplatinum(II) (cis-DDP) to DNA by means of chemical probes. In order to test the chemical probes, experiments were first carried out on two platinated oligonucleotides. It has been verified by circular dichroism and gel electrophoresis that the binding of cis-DDP to an AG or to a GTG site within a double-stranded oligonucleotide distorts the duble helix. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers strongly suggests that the platinated oligonucleotides are bent. The reactivity of the oligonucleotide platinated at the GTG site with chloroacetaldehyde, diethyl pyrocarbonate, and osmium tetraoxide, respectively, suggests a local denaturation of the double helix. The 5''G residue and the T residue within the adduct are no longer paired, while the 3''G residue is paired. The double helix is more distorted (but not denatured) at the 5'' side of the adduct than at the 3'' side. In the case of the oligonucleotide platinated at the AG site, the double helix is also more distorted at the 5'' side of the adduct than at the 3'' side. The G residue within the adduct is paired. The reactivities of the chemical probes with six platinated DNA restriction fragments show that even at a relatively high level of platination only a few base pairs are unpaired but the double helix is largely distorted. No local denaturation has been detected at the GG sites separated from the nearest GG or AG sites by at least three base pairs. The AG sites separated from the nearest AG or GG sites by at least three base pairs do not denature the double helix locally when they are in the sequences puAG/nyTC. When they are in the pyAG/puTC sequences, the reactivity of osmium tetraxide with the T residues complementary to the platinated A residues indicates either a distortion or an unpairing of the bases. The T residues within the sequences (CGT/GCA) react strongly with osmium tetraxoide. It is suggested that the distortion within these sequences is induced by adducts located further away along the DNA fragments, these sequences not being the major sites for the binding of cis-DDP.