Oxidatively Induced DNA−Protein Cross-Linking between Single-Stranded Binding Protein and Oligodeoxynucleotides Containing 8-Oxo-7,8-dihydro-2‘-deoxyguanosine

Abstract

The formation of covalent cross-links between amino acid side chains and DNA bases in DNA-protein complexes is a significant pathway in oxidative damage to the genome, yet much remains to be learned about their chemical structures and mechanisms of formation. In the present study, DNA-protein cross-links (DPCs) were formed between synthetic oligodeoxynucleotides containing an 8-oxo-7,8-dihydro-2'deoxyguanosine (OG) or an 8-oxo-7,8-dihydro-2'-deoxyadenosine (OA) nucleotide and Escherichia coli singled-stranded binding protein (SSB) under oxidative conditions. Studies with various sequences indicated that DNA homopolymers and those lacking 8-oxopurines were less reactive toward DPC formation. DPCs were formed in the presence of HOCl, peroxynitrite, and the one-electron oxidants Na(2)IrCl(6), Na(2)IrBr(6), and Na(3)Fe(CN)(6). Protein-protein cross-linking was also observed, particularly for oxidants of high reduction potential such as Na(2)IrCl(6). The adducted oligodeoxynucleotides were sensitive to hot piperidine treatment leading to strand scission at the site of cross-linking. In addition, the covalent cross-links were somewhat heat and acid labile, which may be related to the difficulties encountered in obtaining complete characterization of trypsin digests of the DPCs. However, model reactions involving the single amino acids lysine, arginine, and tyrosine, residues known to be involved in base contacts in the DNA:SSB complex, could be studied, and the adduct formed between N(alpha)-acetyllysine methyl ester and an 18-mer containing OG was tentatively characterized by electrospray ionization mass spectrometry as analogues of spiroiminodihydantoin and guanidinohydantoin. A mechanism involving nucleophilic attack of an amino acid side chain (e.g. the epsilon-amino group of lysine) at C5 of a 2-electron oxidized form of OG is proposed.