Peroxynitrite-induced DNA damage in the supF gene: correlation with the mutational spectrum

Abstract

Tissue inflammation and chronic infection lead to the overproduction of nitric oxide and superoxide. These two species rapidly combine to yield peroxynitrite (ONOO −), a powerful oxidizing and nitrating agent that is thought be involved in both cell death and an increased cancer risk observed for inflamed tissues. ONOO − has been shown to induce single-strand breaks and base damage in DNA and is mutagenic in the supF gene, inducing primarily G to T transversions clustered at the 5′ end of the gene. The mutagenicity of ONOO − is believed to result from chemical modifications at guanine nucleobases leading to miscoding DNA lesions. In the present work, we applied a combination of molecular and analytical techniques in an attempt to identify biologically important DNA modifications induced by ONOO −. pUC19 plasmid treated with ONOO − contained single-strand breaks resulting from direct sugar damage at the DNA backbone, as well as abasic sites and nucleobase modifications repaired by Fpg glycosylase. The presence of carbon dioxide in the reaction mixture shifted the ONOO − reactivity towards reactions at nucleobases, while suppressing the oxidation of deoxyribose. To further study the chemistry of the ONOO − interactions with DNA, synthetic oligonucleotides representing the mutation-prone region of the supF gene were treated with ONOO −, and the products were analyzed by liquid chromatography–negative ion electrospray ionization mass spectrometry (LC–ESI − MS) and tandem mass spectrometry. 8-Nitroguanine (8-nitro-G) was formed in ONOO −-treated oligonucleotides in a dose-dependent manner with a maximum at a ratio of [ONOO −]: [DNA]=10 and a decline at higher ONOO − concentrations, suggesting further reactions of 8-nitro-G with ONOO −. 8-Nitro-G was spontaneously released from oligonucleotides ( t 1/2=1 h at 37°C) and, when present in DNA, was not recognized by Fpg glycosylase. To obtain more detailed information on ONOO −–induced DNA damage, a restriction fragment from the pSP189 plasmid containing the supF gene (135 base pairs) was [ 32P]-end-labeled and treated with ONOO −. PAGE analysis of the products revealed sequence-specific lesions at guanine nucleobases, including the sites of mutational “hotspots.” These lesions were repaired by Fpg glycosylase and cleaved by hot piperidine treatment, but they were resistant to depurination at 90°C. Since 8-nitro-G is subject to spontaneous depurination, and 8-oxo-guanine is not efficiently cleaved by piperidine, these results suggest that alternative DNA lesion(s) contribute to ONOO − mutagenicity. Further investigation of the identities of DNA modifications responsible for the adverse biological effects of ONOO − is underway in our laboratory.