This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.
DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.
The comet assay is a gel electrophoresis method used to detect DNA breaks at the single-cell level. It relies on the principle that open-ended DNA breaks migrate under electrophoretic conditions, while intact DNA remains largely static. Broken DNA migrates because breaks result in the relaxation of supercoiled DNA, causing its gradual spatial relocation towards the anode in the electrophoretic chamber, which results in a comet-like appearance observed by immunofluorescence1,2. The extent of DNA damage is then measured by quantifying the amount of broken DNA that has migrated relative to the compact DNA.
The two most commonly used comet assay methods are the alkaline comet (AC) assay and the neutral comet (NC) assay. The AC assay is performed under denaturing conditions using a high alkaline pH solution, while the NC assay is carried out in a solution with neutral pH. Both NC and AC assays can reliably detect DNA breaks that occur in the nucleus. However, the alkaline version is also advantageous for detecting alkali-labile sites3. Both formats of the assay require the use of lysis conditions to ensure that DNA is free of proteins prior to electrophoresis.
This assay is a simple method for detecting DNA breaks and offers several unique advantages for determining cellular DNA damage. The method is relatively easy to set up and requires reagents that can either be prepared in the laboratory or purchased from commercial vendors. As a single-cell resolution technique, the assay requires very little starting material. Notably, the NC assay can measure breaks with high sensitivity, reported to detect between 50 and 10,000 breaks per cell4. The versatility of this technique is demonstrated by its wide range of applications, including ecotoxicology5, human biomonitoring6, and genotoxicity studies7. The assay can also be reliably used across various cell types and adapted for high-throughput assays8 and for assessing breaks at specific genomic regions9. Thus, this assay serves as a rapid and reliable technique for investigating break formation at the single-cell level.
The process of DNA replication is constantly challenged by several endogenous and exogenous stressors10,11. Stalling of replisomes due to such lesions can cause DNA breaks and result in heightened genome instability. DNA breaks also often arise as replication intermediates to facilitate DNA repair12. Additionally, several chemotherapeutic agents induce replication-dependent double-strand breaks (DSBs), such as camptothecin (CPT)13,14,15and PARP inhibitors16,17. Thus, this assay serves as a powerful methodology to study the nature of DNA lesions, assess the processing of replication forks, and investigate the therapeutic potential of DNA-damaging agents. Adaptations of the assay that involve labeling newly synthesized DNA with BrdU have been useful for studying replication-associated stress phenotypes18,19,20,21. Notably, the NC assay is a reliable method for studying DNA break induction in the context of replication, as demonstrated in studies involving the characterization of fork proteins22,23, analysis of replication intermediates24,25, and investigation of transcription-replication conflicts in genome maintenance26,27. Herein, we outline a NC assay protocol with the goal of quantifying DNA breaks during replication in human cells. This protocol can be readily implemented by researchers interested in assessing replication stress-associated damage in a wide variety of dividing human cells.
This protocol (Figure 1) primarily utilizes adherent U2OS cancer cell lines but is adaptable to a wide variety of adherent and suspension cells grown in tissue culture. The solutions and buffers used in this study are detailed in Table 1. The reagents and equipment used are listed in the Table of Materials.
1. Preparation of materials
2. Preparation of samples
3. Cell resuspension and lysis
4. Electrophoresis
5. Staining
6. Imaging and analyses
Analysis of break induction by replication stress reagents
U2OS cells were treated with DMSO, 4 of mM hydroxyurea (HU), or 100 nM of CPT for 4 h and analyzed for break accumulation by the NC assay (Figure 6A). While CPT induces breaks during S-phase by blocking topoisomerase-113, HU-induced depletion of nucleotide pools stalls replication forks that are progressively converted into DSBs28. The data indicates that exposure to either HU or CPT triggers break induction in S-phase, which is detectable by NC assay.
Analysis of break induction comparing lysis at different temperatures
Untreated U2OS cells were incubated in lysis buffer either at room temperature (RT) or 4 °C prior to analyses of break induction (Figure 6B). The results indicate that the temperature at which lysis is performed has minimal impact on the migration of tails in the assay.
Temporal analysis of breaks by hydroxyurea (HU)
U2OS cells were left either untreated or treated with 4 mM of HU for 4 h and 8 h prior to analyses of break induction by the NC assay (Figure 7). The results indicate that prolonged stalling of replication forks with HU causes replication fork collapse into DSBs.
Dose-dependent analysis of breaks by camptothecin
U2OS cells were treated with increased doses of CPT for 1 h and harvested for break analyses using the NC assay (Figure 8). Whereas break accumulation is minimal in cells not treated with CPT, the degree of break induction gradually increases with increased dosage of CPT, thus demonstrating the utility of the assay in studying dose-dependent effects on break accumulation during DNA replication.
Comparative analysis of breaks across multiple cell types
Basal levels of DNA breaks were assessed across three divergent mitotic cell types- hTERT immortalized retinal pigment epithelial cells (hTERT-RPE-1), simian virus 40 (SV40) large T-antigen expressing human embryonic kidney cells (HEK293T) and bone osteosarcoma cells (U2OS) (Figure 9). Each cell line demonstrated differential migration of tail moments in the absence of added replication stress, which may be due to differences in cell cycle distribution, chromatin organization, or number of active replication forks. Each cell type was also exposed to 1 µM of CPT for 1 h to validate an increase in replication-dependent DNA breaks.
Analyses of break induction by AC assay
U2OS cells were treated with DMSO, 4 mM of HU, 1 µM of CPT, or 10 µM of PARP inhibitor (PARPi) Olaparib and analyzed for break induction (Figure 10). Cells were treated with DMSO, HU, and PARPi for 2 h while CPT treatment was for 1 h. The results indicate that exposure to replication stress causes break accumulation, which is observable by the AC assay.
Figure 1: Schematic representation of the NC assay. (A) The required number of cells is seeded the day before in a 6-well dish. (B) After drug treatments, the cells are trypsinized and harvested in cold PBS. (C) The cells are gently resuspended in melted agarose at a ratio of 10:1 (10 µL of agarose for 1 µL of cell suspension) using a pipette tip. (D) The cell suspension is transferred onto a slide well using a pipette tip and spread uniformly to cover the well surface completely with agarose. (E) Following incubation at 4 °C, the slides are immersed in lysis buffer, followed by cold 1x TAE buffer. (F) The samples are subjected to electrophoresis using the appropriate apparatus. (G) The slides are then immersed in the DNA precipitation solution, followed by a 70% ethanol solution. (H) After drying, the wells are stained with 1x SYBR Green solution. (I) The slides are imaged using an epifluorescence microscope. Please click here to view a larger version of this figure.
Figure 2: Side view of agarose on comet slide wells. (A) After incubation at 4 °C (step 3.8), the solidified agarose appears as a 'bubble' with a convex shape. (B) After drying at 45 °C (step 5.3), the agarose appears flattened on the well. Please click here to view a larger version of this figure.
Figure 3: Imaging and analysis of comets. (A) Comets within the inner circle of the well (highlighted in blue) are selected for analysis. Comets toward the outer edge of the well are excluded due to atypically longer tail moments, marking them as outliers. (B) At least 100 representative comets are randomly selected from the blue-colored region. Please click here to view a larger version of this figure.
Figure 4: Representative image of comets obtained from the NC assay. The right panel displays a representative field of view for SYBR Green-stained nuclei, while the left panel highlights the region (green outline) used for scoring comets. Nuclei that are partially visible or stacked close to each other (red outline, right panel) are not included in the analysis. Comets selected for scoring should be individually distinguishable, fully contained within the field of view, and exhibit uniform staining intensity. Please click here to view a larger version of this figure.
Figure 5: Representative scoring of comets obtained from the NC assay. (A) Individual nuclei are outlined using CometScore software. (B) The full spectrum view (left panel) is enabled, and the threshold (cutoff bar) is adjusted to achieve optimal signal intensity with minimal background. The 'full spectrum' signal should correspond to the 'single hue' image (top image, right panel) and not exceed the outline (bottom image, right panel). (C) The vertical yellow line in the box is aligned with the center of the comet head, using the widest part of the comet head as a reference for consistency. (D) The score for each analyzed comet is reported in the upper right corner. Please click here to view a larger version of this figure.
Figure 6: Analysis of break induction by replication stress reagents. (A) U2OS cells were treated with DMSO, 100 nM camptothecin (CPT), or 4 mM hydroxyurea (HU) before break induction analysis. The representative graph shows increased break accumulation in cells treated with CPT or HU. (B) Untreated U2OS cells were seeded on comet slides and incubated for lysis (step 3.9) either at room temperature (RT) or 4 °C to compare the effect of lysis temperature. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.
Figure 7: Temporal analysis of breaks induced by hydroxyurea. U2OS cells were either untreated or treated with 4 mM HU for 4 h or 8 h before break induction analysis. The representative graph depicts a gradual increase in break accumulation in cells treated with HU. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.
Figure 8: Dose-dependent analysis of breaks induced by camptothecin. U2OS cells were treated with either DMSO or increasing concentrations of CPT (25 nM, 100 nM, 500 nM, and 1 µM) for 1 h before break analysis. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. The representative graph shows a dose-dependent increase in breaks induced by CPT. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.
Figure 9: Comparative analysis of breaks across multiple cell types. hTERT-RPE-1, HEK293T, and U2OS cells were treated with either DMSO or 1 µM CPT for 1 h. The representative graph shows the relative extent of DNA damage across the three cell types in the presence or absence of CPT. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.
Figure 10: Analysis of break induction by the AC assay. U2OS cells were treated with DMSO, 4 mM HU, 1 µM CPT, or 10 µM PARP inhibitor Olaparib prior to break induction analysis. Cells were treated with DMSO, HU, and PARP inhibitor for 2 h, while CPT treatment was for 1 h. The representative graph demonstrates increased break accumulation in cells treated with CPT, HU, or PARP inhibitors. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.
Reagent | Working solution |
Lysis Buffer | 0.5% SDS, 200 mM Tris-Cl (pH 7.4), 50 mM EDTA |
1x TAE Buffer | 40 mM Tris Base, 20 mM Acetic Acid, 1 mM EDTA in pH 8.45 |
DNA Precipitation solution | 1 M Ammonium Acetate, 87% Ethanol |
70% Ethanol solution | 70% Ethanol in distilled water |
1x SYBR Green solution | 1x SYBR Green in 1x TAE buffer |
DMEM culture media | DMEM with 7.5% FBS |
DMEM/F12 culture media | DMEM/F12 with 7.5% FBS |
Alkaline unwinding solution (for use in Alkaline Comet Assay) | 200 mM NaOH, 1 mM EDTA in pH>13 |
Table 1: List of buffers and solutions.
This protocol outlines a NC assay for assessing DNA breaks in human cells undergoing active replication. The protocol is relatively simple to perform, and researchers can readily adapt it to high pH conditions for alkaline analyses8. It includes two critical steps, as described in steps 3.7 and 4.5. In step 3.7, it is essential to spread the melted agarose with cells uniformly across the well to prevent comets from overlapping during analysis. Ensure that cellular aggregates are dissolved before resuspending the cells in agarose. Preventing the release of the pipette tip while spreading the agarose will help minimize the introduction of bubbles. The conditions for electrophoresis are crucial for achieving appropriate comet tail migration for imaging and analysis. Researchers should consider the nature of the cell lines used for analysis, as the extent of tail migration and the percentage of cells exhibiting comet tails vary across cell types (Figure 9). Therefore, it is recommended to empirically determine the electrophoresis run time prior to experimentation.
If no comet tails are observed in positive control samples (CPT-treated cells), the drug efficacy should be corroborated in parallel using alternative approaches, such as immunostaining for the DNA damage marker γH2AX. Extensive tail migration can preclude the analysis of break induction. Minimizing electrophoresis time and assessing cell morphology prior to analysis can prevent obtaining comet tails that are too long. Insufficient drying of the slides can result in blurry and hazy comet images. Ensure that the agarose on the comet slide is evenly dried and flattened before staining with 1x SYBR solution. If the range of fold induction is narrow, performing a dose-response curve with CPT can help determine an optimal working range for experimental purposes. Applying these troubleshooting tips will help maximize the results from these assays.
This assay methodology is cost-effective and highly sensitive compared to other techniques used to study DNA break induction, such as the Halo assay29,30, TUNEL assay31, and pulsed-field gel electrophoresis32. However, the current assay technique has certain limitations. It does not provide a precise number of DNA lesions but offers a relative estimation of break accumulation compared to undamaged cells. Additionally, it cannot assess break induction that occurs in a cell cycle-dependent manner. To exclude changes in break induction due to differences in cell cycle progression, cell cycle analyses by flow cytometry should be performed in parallel.
The authors have nothing to disclose.
We thank the Dungrawala lab members for their input and feedback. This work was funded by NIH grant R35GM137800 to HD.
1x DPBS | Gibco | 14190144 | |
Camptothecin | Selleckchem | S1288 | |
Comet LMAgarose (LMA) | R&D systems | 4250-050-02 | |
CometAssay Electrophoresis System II | R&D systems | 4250-050-ES | Includes electrophoresis tank, safety lid, cables, 2/20 wells slide trays and slide tray overlay |
CometScore software | TriTek | open-sourced | |
CometSlide | R&D systems | 4250-050-03 | |
DMEM, high glucose | Gibco | 11965092 | |
DMEM/F12 | Gibco | 11320033 | |
DMSO | FisherSci | D128-4 | |
Epifluorescence microscope | Keyence | BZ-X810 | |
Fetal Bovine Serum – Premium | Bio-Techne | S11150 | |
Gibco Trypsin-EDTA (0.05%), phenol red | Gibco | 25300054 | |
GraphPad Prism 10.0 | GraphPad | ||
HEK293T cells | ATCC | CRL-11268 | |
hTERT-RPE-1 cells | ATCC | CRL-4000 | |
Hydroxyurea | Millipore Sigma | H8627 | |
PARP inhibitor Olaparib | Selleckchem | S8096 | |
PowerPac | FisherSci | FB300Q | |
Surface Treated Sterile Tissue Culture Plate | FisherSci | FB012927 | |
SYBR Green I Nucleic Acid Gel Stain (10,000x) | FisherSci | S7567 | |
U2OS cells | ATCC | HTB-96 | |
UVP HB-1000 Hybridization Incubator | FisherSci | UVP95003001 |