Elsevier

DNA Repair

Volumes 66–67, June–July 2018, Pages 42-49
DNA Repair

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

https://doi.org/10.1016/j.dnarep.2018.04.007Get rights and content

Highlights

  • 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.

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.

Graphical abstract

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.

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Introduction

Nucleic acids are continuously subjected to modification by endogenous and exogenous sources. The formation and retention of these nucleic acid modifications or adducts can threaten the fidelity of the genome by altering the nucleic acid structure, changing base pairing and promoting the likelihood of insertions, deletions and translocations. Detection and removal of DNA damage is essential for maintaining genomic integrity and a tailored and lesion specific DNA damage response (DDR) has evolved for signaling the enzymatic recognition of DNA adducts and corrdinating repair by a suite of DNA repair pathways. Mutations in genes involved in DNA repair are linked to aging and genetic diseases, as well as cancer predisposition, and these mutations can also alter treatment outcomes [[1], [2], [3], [4]]. Therefore, assessment of DNA damage formation and persistence in cells aids in the determination of the genotoxic or carcinogenic potential of chemical or environmental exposures and may identify subpopulations vulnerable to exposure effects. This potential has led to the development of assays that monitor and measure the formation and retention of DNA adducts within a genome, in order to assess the functional DNA repair capacity (reviewed in [[5], [6], [7]]).

Liquid chromatography and mass spectrometry have been used extensively to identify and quantify DNA adducts. These methods have allowed precise quantitation of adduct levels in purified DNA samples and have significantly advanced our understanding of the structure and lifetime of DNA adducts. However, these techniques require expert users, expensive equipment, often employ isotopic labeling for precise quantitation, and require microgram quantities of isolated DNA [6,8,9]. While there are distinct advantages to utilizing these techniques to measure specific adducts, there are issues with DNA isolation procedures introducing further DNA damage and in the standardization of measurements [10].

More accessible forms of DNA damage and adduct detection are antibody based strategies, comet assays, and enzymatic detection by terminal deoxynucleotidyl transferase (TdT). Antibody strategies can be applied to isolated DNA, in cells, or in fixed tissues. While antibodies exist for strand break signals (γH2AX or 53BP-1) and some DNA lesions (6-4 photoproducts, cyclobutane pyrimidine dimers (CPD), etc.), these techniques are limited by the variation and specificity of available antibodies and may be difficult to multiplex due to incompatibilities in fixation or staining procedures. Comet assay or Single Cell Gel Electrophoresis allows more specific strand break detection in cells, eliminating the requirements for specific antibodies, and with modifications can detect alkali labile sites, oxidative base damage, and DNA cross-linking [11,12]. However, comet assay has been difficult to standardize and reproduce from lab to lab, though comet chip technologies and automated image processes are improving these shortcomings [[13], [14], [15]].

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and in situ (DNA) end labeling (ISEL) [[16], [17], [18]] have also been employed extensively over the past 20 years to detect DNA strand breaks during apoptosis and in some cases DNA damage across a variety of biological samples [[19], [20], [21]]. However, just like the other methods, there are drawbacks to using TUNEL or ISEL because they are highly specific for 3′-OH ends. Several TUNEL modifications have emerged extending its ability to detect other types DNA ends (i.e., 3′-PO4) or improve DNA damage detection by incorporating FPG to excise oxidative DNA adducts [22].

While all of these techniques are used extensively in the literature to assess DNA damage and adduct formation, each has significant limitations for broad spectrum detection of DNA damage. This gap in methodologies has led us to develop the Repair Assisted Damage Detection (RADD) assay, which harness the action of specific DNA repair enzymes to recognize and excise DNA adducts throughout the genome. Once the DNA adduct has been removed, the adduct position is tagged by insertion of a biotinylated deoxyuridine triphosphate (dUTP). This method has proven viable for detecting DNA lesions on isolated DNA [23], and here we demonstrate for the first time that this detection scheme can be extended to fixed cells to measure DNA damage in situ.

The assay provides a novel platform for the characterization of nuclear DNA damage within and across different cell lines by scoring the DNA lesion load. The experiments outlined herein demonstrate that RADD is a robust and novel assay for the measure of nuclear DNA damage and has the potential to be used to investigate specific DNA repair mechanisms, to address risk assessment for both environmental toxicology and cancer etiology, and to evaluate DNA targeted cancer therapies.

Section snippets

Cell culture

A375P cells were purchased from the American Type Culture Collection (ATCC CRL-3224) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Hyclone #SH30022.01) and supplemented with 10% fetal bovine serum (Atlanta Biologicals # S11550) and 1% sodium pyruvate (Gibco # 11360-070). Chinese hamster ovary (CHO-K1) cells were received from Dr. Samuel H. Wilson at the National Institute of Environmental Health Sciences and grown in minimal essential medium (MEM, Hyclone #SH30265FS)

RADD theory and reaction components

The RADD assay was designed as an adaptable assay to characterize a broad spectrum of DNA adducts in fixed cells. Permeabilization allows the RADD enzymes access to the nucleus in order to process the DNA lesions present in the nuclear DNA. DNA lesions are first processed by a well characterized suite of DNA repair enzymes, and the resulting gaps are then filled with a biotinylated nucleotide to allow for the DNA damage to be scored by fluorescence microscopy (Fig. 1).

The RADD assay utilizes

Discussion

The dynamic nature of DNA damage and repair requires creative methods to characterize, quantify, and localize DNA adducts. New methodologies are needed to bridge the gap between detecting single DNA adducts with extreme precision and detecting large numbers of adducts in a single method. As demonstrated here the RADD assay provides a dynamic, simple and accessible technique to assess a broad spectrum of DNA adducts in a cellular context over time and is capable of measuring global nuclear DNA

Conclusion

RADD is able to detect nuclear DNA lesions in cells and can detect damage across species and tissue types. The RADD assay is highly adaptable and includes: (1) a DNA damage processing mix containing DNA repair enzymes that recognize, remove, and modify DNA lesion sites to contain the appropriate DNA end chemistry for gap filling, and (2) a gap filling mix containing a tagged nucleotide to be incorporated for monitoring the processed DNA damage site. The DNA repair enzymes that are utilized by

Conflict of interest statement

The authors declare that there are no conflicts of interest

Acknowledgments

The Gassman lab is supported by start-up funding from the University of South Alabama Mitchell Cancer Institute. The Ebenstein lab acknowledges funding from the BeyondSeq consortium [EC program 63489]. The authors would like to thank Dr. Joel Andrews and the Cellular and Biomolecular Imaging Facility for assistance with confocal microscopy experiments. This work was supported by R21ES028015 from National Institutes of Health, National Institute of Environmental Health Sciences.

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  • Cited by (17)

    • DNA damage measurements within tissue samples with Repair Assisted Damage Detection (RADD)

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      Detection of the DNA damage occurs by combing and imaging the isolated repaired DNA or by imaging the fluorescent content of repaired cells with confocal microscopy or flow cytometry. We have utilized this system to monitor the induction of oxidized adducts and ultraviolet (UV) induced DNA adducts within cells and isolated DNA (Holton et al., 2018; Torchinsky et al., 2019). We have also demonstrated the ability of the method to monitor the dynamics of DNA repair by inducing DNA damage and measure DNA adduct levels over time (Holton et al., 2018).

    • Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies

      2019, Current Opinion in Biotechnology
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      Recently, a further modification of this assay, Repair Assisted Damage Detection (RADD) was developed with a refined enzyme cocktail for the detection of abasic sites, uracils, strand breaks, and oxidative and UV-induced DNA lesions within cells (Figure 2) [31••]. Although the RADD in cell assay allows relative quantification of DNA damage [31••], the DNA combing strategy can detect very low levels of endogenous DNA damage 0–10 labels per Mbp and significant increases ∼1–1.5 fold in damage after genotoxic exposures [27••,28•,29,30••]. Additionally, since the DNA context is preserved and damage sites are labeled, these techniques are compatible with optical sequence mapping [27••,28•,29] or next generation sequencing of isolated damage fragments (similar to [33•,34•]), allowing new insight into genome damage susceptibility.

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