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

Abstract

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.

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

“…Based on the altered protein expression and localization of BER proteins in TNBC cells, we evaluated cells for variation in levels of basal DNA damage using the broad spectrum DNA damage assay, Repair Assisted Damage Detection (RADD) [30]. RADD uses bacterial DNA glycosylases to detect and excise abasic sites, oxidative base lesions, bulky DNA adducts, and thymine dimers (see Material and Methods).…”

“…Then the gaps in the DNA where the lesions were excised and any strand breaks occur are then tagged by Klenow polymerase with a Digoxigenin-11-dUTP. Damage sites are detected by immunofluorescence as described in the materials and methods [30]. RADD was applied to the TNBC cell panel, and the nuclear fluorescence intensity of basal DNA damage quantified (Fig 5, S3 Fig).…”

“…Cells were plated in 8-well chambered coverglass, fixed, and permeabilized using the same procedure as for immunofluorescence. The Repair Assisted Damage Detection (RADD) assay was performed as described previously with minor modifications [30]. After permeabilization, cells were incubated with uracil DNA glycosylase (UDG) to remove uracil (NEB #M0304S), formamidopyrimidine [Fapy]-DNA glycosylase (Fapy-DNA glycosylase NEB #M0240S) to remove Fapy lesions, T4 Pyrimidine dimer glycosylase (T4PDG NEB #M-308S) to remove pyrimidine dimer lesions, endonuclease IV (Endo IV NEB #M0304S) to process oxidative damage, AP sites and modifies 3’ phosphates to 3’ OH, and endonuclease VIII (Endo VIII NEB #M0299S) to remove damaged pyrimidines diluted in 1X Thermpol buffer and incubated at 37 degrees C for 1 hour.…”

“…Samples were then counterstained with Alexa Fluor goat anti-mouse 546 at 1:400 in 2% BSA in PBS for 45 min at room temperature and nuclei were stained as for immunofluorescence. Image analysis was performed as described previously [30]. MDA-231 cells were used to set the 546 nm laser gain, and the other cells were imaged at that gain.…”

Measuring biological aging in humans: A quest

“…While several biochemical and cell‐based tests of DNA repair capacity have been developed and shown to be reasonably objective and reliable, quantification of DNA repair capacity in humans remains unsatisfactory (Berwick & Vineis, ; Trzeciak et al, ; Trzeciak, Barnes, & Evans, ). The few tests described in the literature have not been applied to large populations and lack independent validation (El‐Zein et al, ; Fang, Neutzner, Turtschi, Flammer, & Mozaffarieh, ; Hamann & Hartwig, ; Holton, Ebenstein, & Gassman, ; Nagel et al, ; Reddy et al, ). Moreover, there is no consensus on gold standard assays and most methods require large amounts of freshly collected pure cell types, and these only address repair capacity of a subset of specific lesions.…”