Formation of DNA-protein cross-links involving the initial formation of a guanine radical cation was investigated. For this purpose, riboflavin-mediated photosensitization of a TGT oligonucleotide in aerated aqueous solution in the presence of the KKK tripeptide was performed. We have shown that the nucleophilic addition of the epsilon-amino group of the central lysine residue of KKK to the C8 atom of either the guanine radical cation or its deprotonated form gives rise to the efficient formation of a Nepsilon-(guanin-8-yl)-lysine cross-link. Interestingly, the time course of formation of the above-mentioned cross-link was found to be not linear with the time of irradiation, and its formation rapidly reached a plateau. This is explained by secondary decomposition of the initially generated cross-link which could be further oxidized more efficiently than starting TGT oligonucleotide. One-electron oxidation of the initially generated cross-link was found to produce mainly two diastereomeric cross-links exhibiting a spiroimino-trilysine-dihydantoin structure as inferred from enzymatic digestion, CD, UV, NMR and mass spectrometry measurements. In addition, other minor cross-links, for which formation was favored at acidic pH, were assigned as lysine-guanine adducts in which the modified guanine base exhibits a guanidino-trilysine-iminohydantoin structure. A proposed mechanism for the formation of the different detected oligonucleotide-peptide cross-links is given. The high yield of formation of the detected cross-links strongly suggests that a DNA-protein cross-link involving a lysine residue linked to the C8 position of guanine could be generated in cellular systems if a lysine is located in the close vicinity of a guanine radical cation.

Copper is an important biological metal that tightly binds to DNA. Its reaction with endogenously generated hydrogen peroxide may thus lead to the formation of DNA damage. To gain insights into the underlying mechanisms, a comparative study of the damage produced within isolated DNA upon exposure to gamma-radiation in aqueous solution, a source of hydroxyl radicals, and incubation with Cu(I) or Cu(II) complexes in the presence of hydrogen peroxide was carried out. Several relevant base modifications were quantified by HPLC-tandem mass spectrometry. It was first shown that addition of copper ions only slightly modified the profile of radiation-induced lesions within DNA. However, the distribution of base modifications was drastically different upon incubation of DNA with Cu(I) or Cu(II) complexes in the presence of H(2)O(2). Indeed, guanine degradation products were produced in much higher yield than lesions of the other bases. These observations are rationalized in terms of the occurrence of one electron oxidation with Cu(I) complexes, as confirmed by the study of the degradation of free thymidine. In contrast, the formation of the sole 8-oxo-7,8-dihydroguanine upon incubation of DNA with Cu(II) ions and H(2)O(2) strongly suggests the production of singlet oxygen as the predominant reactive oxygen species.