Glycosidase-Enabled Locus-Specific Detection of 8-Oxo-7,8-dihydroguanine in Genomes under Oxidative Stress

Abstract

Oxidative DNA damage is closely linked to the onset of various age-related diseases. A significant oxidation product, 8-oxo-7,8-dihydroguanine (8OG), has been considered an important epigenetic-like marker in regulating gene expression. Accurately quantifying the locus-specific 8OG levels is crucial for understanding its functional roles in disease induction and gene regulation. In this study, we developed a glycosylase cleavage-mediated extension stalling (GCES) method for the locus-specific detection and quantification of 8OG in genomic DNA. This method utilizes the 8OG-DNA glycosylase Pab-AGOG, which induces single-strand breaks in DNA containing 8OG. The resulting cleavage is then assessed and quantified by using quantitative real-time PCR (qPCR). We successfully applied this strategy to evaluate locus-specific 8OG in synthesized DNA and genomic DNA from HEK293T, HeLa, and HepG2 cell lines under oxidative stress or triclosan treatment. The results demonstrate that the GCES method is accurate and suitable for the site-specific quantification of 8OG in both synthesized and genomic DNA from biological samples. We observed an increased level of 8OG in genomic DNA from biological samples treated with H₂O₂ or triclosan, indicating that both agents can elevate 8OG level in DNA. Overall, the GCES method provides a valuable, straightforward, and cost-effective tool for the quantitative detection of 8OG in DNA at base resolution, which facilitates the investigation of its functional roles as an epigenetic-like or DNA damage marker.