Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD)

• Published in: DNA repair 2018-06, Vol.66-67, p.42-49
• Main Authors: Holton, Nathaniel W., Ebenstein, Yuval, Gassman, Natalie R.
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; Confocal microscopy; DNA adduct; DNA Adducts - analysis; DNA Adducts - metabolism; DNA damage detection; DNA Repair; Environmental Exposure - adverse effects; Fluorescence; Humans; Mutagenicity Tests - methods
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2018.04.007

The Repair Assisted Damage Detection assay is a modular cell based assay for the detection of basal DNA damage and DNA damage induced by exogenous exposures. [Display omitted] •RADD is a streamlined assay for monitoring DNA repair dynamics without DNA isolation.•Enzymatic cocktails allow broad spectrum DNA damage detection in cells.•Cocktails can be easily tailored by user for specific adduct detection, if desired.•Fluorescent read out compatible with high content screening or imaging techniques. 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.