Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD)
Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD)
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DOI：
10.1016/j.dnarep.2018.04.007
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发表时间：
2018-06-01
期刊：
影响因子：
3.8
通讯作者：
Gassman, Natalie R.
中科院分区：
文献类型：
--
作者：
Holton, Nathaniel W.;Ebenstein, Yuval;Gassman, Natalie R.
环境暴露、细胞代谢的反应性副产物和自发脱氨事件会导致一系列DNA加合物，如果这些加合物没有得到修复，就会通过诱导突变、增加不稳定性和促进癌症的发生和发展来威胁基因组的完整性。细胞和组织中DNA加合物的评估对于化学暴露的遗传毒性和致癌性评估至关重要，并可能提供对癌症病因的见解。已经开发了许多方法来表征DNA加合物的形成及其保留以进行风险评估。然而，这些方法的实施和广泛使用仍然存在显着的缺点，因为它们通常需要大量的生物样品，高度专业化的专业知识和设备，并且取决于技术，一次可能仅限于少量DNA加合物的检测和定量。目前迫切需要高通量、易于实施的分析方法，以评估广泛的DNA损伤，从而更快地评估化学暴露和评估生物样品中加合物的保留。在这里，我们描述了一种新的方法，修复辅助损伤检测（RADD），它利用DNA损伤处理修复酶鸡尾酒来检测和修饰DNA损伤位点，以便随后进行标记DNA损伤位点的间隙填充反应。这种检测和标记细胞内广泛DNA损伤的能力，为评估暴露于环境因子或DNA修复能力自然变化的细胞中的DNA损伤水平提供了一种新颖且易于使用的工具。
Environmental exposures, reactive by-products of cellular metabolism, and spontaneous deamination events result in a spectrum of DNA adducts that if un-repaired threaten genomic integrity by inducing mutations, increasing instability, and contributing to the initiation and progression of cancer. Assessment of DNA adducts in cells and tissues is critical for genotoxic and carcinogenic evaluation of chemical exposure and may provide insight into the etiology of cancer. Numerous methods to characterize the formation of DNA adducts and their retention for risk assessment have been developed. However, there are still significant drawbacks to the implementation and wide-spread use of these methods, because they often require a substantial amount of biological sample, highly specialized expertise and equipment, and depending on technique, may be limited to the detection and quantification of only a handful of DNA adducts at a time. There is a pressing need for high throughput, easy to implement assays that can assess a broad spectrum of DNA lesions, allowing for faster evaluation of chemical exposures and assessment of the retention of adducts in biological samples. Here, we describe a new methodology, Repair Assisted Damage Detection (RADD), which utilizes a DNA damage processing repair enzyme cocktail to detect and modify sites of DNA damage for a subsequent gap filling reaction that labels the DNA damage sites. This ability to detect and label a broad spectrum of DNA lesions within cells, offers a novel and easy to use tool for assessing levels of DNA damage in cells that have been exposed to environmental agents or have natural variations in DNA repair capacity.