Ji Tong-Tong, Wang Min, Guo Xia, Gang Fang-Yin, Zhang Shan, Xiong Jun, Gu Yao-Hua, Xie Neng-Bin, Yuan Bi-Feng
Department of Occupational and Environmental Health, School of Public Health, Wuhan University, Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
Research Center of Public Health, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Anal Chem. 2025 Apr 22;97(15):8564-8573. doi: 10.1021/acs.analchem.5c00632. Epub 2025 Apr 12.
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 HO 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.
氧化DNA损伤与多种年龄相关疾病的发生密切相关。一种重要的氧化产物，8-氧代-7,8-二氢鸟嘌呤（8OG），被认为是调节基因表达的重要表观遗传样标记。准确量化位点特异性的8OG水平对于理解其在疾病诱导和基因调控中的功能作用至关重要。在本研究中，我们开发了一种糖基化酶切割介导的延伸停滞（GCES）方法，用于在基因组DNA中对8OG进行位点特异性检测和定量。该方法利用8OG-DNA糖基化酶Pab-AGOG，它能在含有8OG的DNA中诱导单链断裂。然后通过定量实时PCR（qPCR）对产生的切割进行评估和定量。我们成功地应用了这一策略，在氧化应激或三氯生处理下，评估了来自HEK293T、HeLa和HepG2细胞系的合成DNA和基因组DNA中的位点特异性8OG。结果表明，GCES方法准确且适用于对生物样品的合成DNA和基因组DNA中的8OG进行位点特异性定量。我们观察到用HO或三氯生处理的生物样品的基因组DNA中8OG水平升高，表明这两种试剂都能提高DNA中的8OG水平。总体而言，GCES方法为在碱基分辨率下对DNA中的8OG进行定量检测提供了一种有价值、直接且经济高效的工具，这有助于研究其作为表观遗传样或DNA损伤标记的功能作用。
