In Situ Analysis of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Oxidation Reveals Sequence- and Agent-Specific Damage Spectra

Abstract

Guanine is a major target for oxidation in DNA, with 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) as a major product. 8-oxodG is itself significantly more susceptible to oxidation than guanine, with the resulting damage consisting of more than 10 different products. This complexity has hampered efforts to understand the determinants of biologically relevant DNA oxidation chemistry. To address this problem, we have developed a high mass accuracy mass spectrometric method to quantify oxidation products arising site specifically in DNA. We applied this method to quantify the role of sequence context in defining the spectrum of damage products arising from oxidation of 8-oxodG by two oxidants: nitrosoperoxycarbonate (ONOOCO[subscript 2]–), a macrophage-derived chemical mediator of inflammation, and the classical one-electron oxidant, riboflavin-mediated photooxidation. The results reveal the predominance of dehydroguanidinohydantoin (DGh) in 8-oxodG oxidation by both oxidants. While the relative quantities of 8-oxodG oxidation products arising from ONOOCO[subscript 2]– did not vary as a function of sequence context, products of riboflavin-mediated photooxidation of 8-oxodG were highly sequence dependent. Several of the 8-oxodG oxidation products underwent hydrolytic conversion to new products with half-lives of 2–7 h. The results have implications for understanding the chemistry of DNA oxidation and the biological response to the damage, with DNA damage recognition and repair systems faced with a complex and dynamic set of damage targets.