Interstrand crosslinks and sequence specificity in the reaction of cis-dichloro(ethylenediamine)platinum(II) with DNA

Abstract

Characterization of the adducts produced in DNA by the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) and a radiolabeled analogue, [3H]-cis-dichloro(ethylenediamine)platinum(II) ([3H]-cis-DEP) was recently reported (Eastman, A. (1983) Biochemistry 22,3927]. Both drugs reacted at identical sites in DNA, most of which produced intrastrand cross-links. DNA-interstrand cross-links, which represent less than 1% of total platination, have now been characterized. DNA containing interstrand cross-links was enriched for on the basis of its renaturability after boiling. This DNA was digested to deoxyribonucleotides, and the adducts were separated by high-pressure liquid chromatography. A cross-link between two deoxyguanosines was observed to be the most prominent adduct. It is proposed that the major sequence in which this cross-link occurs is 5''-CG-3''. DNA that was incubated with [3H]-cis-DEP for 1 h showed low levels of interstrand cross-links. After removal of unreacted drug, their frequency increased significantly over 6 h with a maximum occurring at about 12 h. A similar phenomenon was seen in the case of intrastrand cross-links that contained adenine, in particular when the cross-link was between the terminal bases in an ANG trinucleotide sequence (N is any nucleotide). The primary site of reaction is at guanine, with a slow subsequent cross-link to the adenine. A model is presented that is consistent with the observation that adenine is always at the 5'' terminus of these adducts. The proportion of adducts at ANG sequences also increased at elevated temperatures. This is discussed with regard to potential significance duriung hyperthermia treatment of patients. Possible distortions in DNA resulting from platination were also investigated. Individual cis-DEP adducts were not recognized by single strand specific S1 nuclease, demonstrating minimal denaturation of the DNA helix. Previous studies showing that these adducts are susceptible to S1 nuclease digestion were performed at high levels of modification, conditions under which the concerted effect of several adducts would have produced a digestible site.