Characterization of Lysine−Guanine Cross-Links upon One-Electron Oxidation of a Guanine-Containing Oligonucleotide in the Presence of a Trilysine Peptide

Abstract

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.