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

Abstract

Detection and characterization of DNA damage is essential for evaluating genotoxicity, monitoring DNA repair, developing biomarkers for exposures, and evaluating the efficacy of chemotherapies. These diverse applications for DNA damage measurements have spurred the continual development and refinement of methodologies for detecting, characterizing, and quantifying DNA damage from isolated DNA and in cells and tissues. Current damage detection methods cover a wide range of techniques from radiolabeling to mass spectrometry, and use of these techniques varies widely based on expense, expertise, and knowledge of adduct formation. More generalizable, easy-to-use methods for detecting and quantifying DNA damage are needed, and there has been an emergence of fluorescence-based methodologies to address this need. Developments in these fluorescence-based strategies are reviewed here.

Applications of CometChip for Environmental Health Studies

“…While these comet-based methods have both been shown to be effective for analysis of DNA damage and repair, other technologies with their own advantages and disadvantages have also been developed and reviewed extensively in the literature. ,− In the remainder of this section, we will briefly highlight a few popular methods discussed in these reviews: The micronucleus assay quantifies chromosomal damage in cells and is a standard test in the regulatory approval process. , This method can be adapted for high-throughput experiments through the use of flow cytometry. , However, compared to the comet assay, the micronucleus assay does not provide information about the types of DNA damage present (e.g., oxidative damage, bulky lesions) or information about DNA repair. An alternative approach for DNA damage analysis is using immunofluorescence methods to measure the extent to which chromatin is modified near a DNA DSB (γ-H2AX staining). − While the comet assay measures a physical break, this method detects a signaling event (the phosphorylation of the H2AX histone), which may be influenced by additional factors. Analytical methods have also been developed that utilize mass spectrometry to accurately measure DNA adducts following exposures, and these studies can provide valuable insight into a chemical’s mechanism of action. − Compared to comet analysis, mass spectrometry is often more expensive and technically challenging. In addition, comet data are relatively straightforward to interpret for new users and provide a simple way to study repair kinetics. Similar to the alkaline comet assay, the Fluorimetric Detection of Alkaline DNA Unwinding (FADU) quantifies DNA strand breaks following DNA unwinding in an alkaline buffer.…”

In Situ Analysis of DNA-Protein Complex Formation upon Radiation-Induced DNA Damage

“…A particular approach in detecting DNA–protein complexes consists of the expression of a fluorescent reporter (i.e., a GFP-labeled peptide or protein) that can bind DNA specifically to a given lesion (e.g., DSB), so that fluorescent localization of DNA damage is obtained in living cells [81]. Expression of the fluorescent GAM protein, or a peptide from 53BP1 protein, have provided localization of DSBs [82,83].…”

Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing

“…An understanding of the repair efficiency and substrate selectivity of different DNA repair enzymes can reveal vulnerabilities in the genome. Second, DNA repair enzymes are potential pharmacological targets where DNA repair pathway inhibition, coupled with other metabolic deficiencies, could result in selective toxicity. − Third, DNA repair enzymes can serve as valuable reagents for characterizing and quantifying specific types of DNA damage, − identifying the location of specific types of DNA damage at nucleotide resolution, − and for preparing input DNA for next-generation sequencing. − …”