Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Summary

The methodology assists in the analysis of variation in DNA replication dynamics in the presence of nanoparticles. Different methodologies can be adopted based on the cytotoxicity level of the material of interest. In addition, a description of the image analysis is provided to help in DNA fiber analysis.

Abstract

Nanomaterial exposure can cause replication stress and genomic instability in cells. The degree of instability depends on the chemistry, size, and concentration of the nanomaterials, the time of exposure, and the exposed cell type. Several established methods have been used to elucidate how endogenous/exogenous agents impact global replication. However, replicon-level assays, such as the DNA fiber assay, are imperative to understand how these agents influence replication initiation, terminations, and replication fork progression. Knowing this allows one to understand better how nanomaterials increase the chances of mutation fixation and genomic instability. We used RAW 264.7 macrophages as model cells to study the replication dynamics under graphene oxide nanoparticle exposure. Here, we demonstrate the basic protocol for the DNA fiber assay, which includes pulse labeling with nucleotide analogs, cell lysis, spreading the pulse-labeled DNA fibers onto slides, fluorescent immunostaining of the nucleotide analogs within the DNA fibers, imaging of the replication intermediates within the DNA fibers using confocal microscopy, and replication intermediate analysis utilizing a computer-assisted scoring and analysis (CASA) software.

Introduction

During each cell cycle, DNA replication ensures accurate genome duplication¹. Eukaryotic chromosomal replication essentially depends on three factors: the timing of the firing of multiple replication origins, the speed of the forks that emerge from the fired origins, and the termination of the replication process when two replication forks from adjacent origins meet². For the high-fidelity transmission of genetic information to daughter cells, as well as the preservation of genetic integrity, accurate DNA replication is crucial. Agents that develop from regular metabolism or are due to artificial or natural environmental materials are constantly attacking the genome. These endogenous and exogenous agents cause replication forks to slow down or stop due to encountering DNA damage caused by these agents, and the forks temporarily slowing down or stopping in response to these difficulties is termed replication stress³. In response to replication stress, cells have developed several molecular pathways that maintain the stability of the disturbed replication forks and allow them to restart⁴. In terms of genetic stability, cell survival, and human disease, these replication stress response mechanisms have emerged as the key factors for maintaining a healthy genome, ensuring cell survival, and decreasing the likelihood of disease formation⁵.

One of the exogenous agents capable of producing replication stress is nanoparticles. Nanoparticles are particles that range in size from 1 nm to 100 nm⁶. Due to their high surface areas, distinctive shapes, and unique chemical properties, nanoparticles are utilized in various medical, pharmaceutical, environmental, and industrial applications^7,8. While nanoparticles have a lot of potential benefits, some of them (due to their inherited nature or longevity) can become toxic. Nanoparticles can also form due to the natural wear and tear of medical implants and be released into the peri-prosthetic region^9,10.

Due to the exposure of humans to a myriad of nanoparticles produced for various applications, research in the field of nanoparticle toxicity has increased tremendously over the past 10 years¹¹. While these research efforts have revealed information in abundance about the potential threat that nanoparticles pose to human health, knowledge about the potential for nanoparticles to cause genotoxicity is still limited. What has been discovered so far is that these nanoparticles can physically interact with the DNA, promote DNA damage, and damage or interfere with the proteins responsible for repairing or replicating DNA¹². To detect how they interfere with DNA replication, DNA fiber combing, radioresistant DNA synthesis (RDS), and DNA fiber analysis are typically used^13,14,15,16.

The DNA fiber combing method is flexible and gives information about replication fork dynamics at the single-molecule level¹⁷. In essence, a salinized coverslip is gently withdrawn from the DNA solution once the DNA ends bind to it. The DNA molecules are straightened and aligned by the solution's meniscus. The homogeneity, spacing, and alignment of the DNA fibers support accurate and dependable fiber tract length measurements. By adjusting the length and sequence of the treatments and the drugs used to cause stress or damage, many aspects of fork advancement can be monitored using this application. In this method, a dual labeling system is used, through which the speed and progression of the replication fork are assessed^17,18. On the other hand, 2D gel electrophoresis takes advantage of the fact that, in agarose gel electrophoresis, branching DNA structures travel more slowly than linear DNA molecules of the same mass, allowing for the clean separation of the two in a 2D run. In fact, this method is investigated to segregate DNA molecules based on their mass in the first run and based on their shape in the second orthogonal run. After genomic DNA fragmentation, the uncommon replication and recombination intermediates develop a branching form, and they may be distinguished from the more common linear molecules in the 2D gel¹⁹.

The RDS method is used for determining how global DNA synthesis is impacted. In this method, the degree of inhibition of global replication is determined by comparing the amount of incorporated radioactively labeled nucleotides, such as [14C] thymidine, in untreated versus treated cells^14,20. The percentage difference in radiolabeling between the untreated and treated cells represents the degree to which the DNA-damaging agent impacts DNA synthesis. Similar to this, another method uses the ability of cells to integrate nucleotide analogs like BrdU (5-bromo-2′-deoxyuridine) for flow cytometry to measure the overall rates of DNA synthesis^21,22. While these methods demonstrate how DNA-damaging agents impact global DNA synthesis, they do not show how individual replicons are affected. Indeed, replicon-level assays are imperative to better understand the initiation and extent of genomic instability in the event of toxic particle (nanomaterial) exposure. DNA fiber autoradiography and electron microscopy are some methods used to determine this^23,24,25,26.

The concepts of replication bubbles and bidirectional replication from unevenly spaced sources were first developed using single-molecule tests like electron microscopy and DNA fiber autoradiography^27,28. The direct observation of branching replication intermediates on specific molecules dispersed across a carbon-coated grid is greatly facilitated by electron microscopy. This method, which is still in use today to track pathological shifts at replication forks, was utilized to locate the first eukaryotic origin of DNA replication²⁸. Fiber autoradiography is centered around the concept of the autoradiographic identification of newly replicated areas and the pulse tagging of chromosomes with tritiated thymidine. The first quantitative evaluation of origin densities and replication fork rates in metazoan genomic sequences was made possible by DNA fiber autoradiography²⁹.

Currently, fiber fluorography methods have taken the place of autoradiography, mainly because fiber fluorography is much faster than autoradiography. In fiber fluorography, two halogenated nucleotide derivatives, such as bromo- (Br), chloro- (Cl), or iododeoxyuridine (IdU), are sequentially incorporated into freshly replicated DNA and then identified by indirect immunofluorescence using antibodies³⁰. Microscopic viewing of the nascent DNA that has incorporated one or both analogs is made possible by immunostaining one of the analogs in one color and the other analog in a different color (e.g., immunostaining nascent DNA with incorporated IdU red and incorporated CldU green) (Figure 1)²¹. Many different types of replication intermediates can be identified by DNA fiber analysis. The most commonly studied are individual elongating forks, initiations, and terminations. Individual elongating forks have a replication pattern of red followed by green (red-green; Figure 2A).¹³ The lengths of these intermediates are frequently used to gauge the fork speed (i.e., fork length/pulse time) or the exonucleolytic degradation of nascent DNA through track shortening (Figure 2E)^30,31,32. In a study by Mimitou et al., it was found that upon long-term exposure to hydroxyurea, a replication poison that causes double-strand breaks in the DNA, RE11 was recruited³³. MRE11 is an exonuclease known for its 3'-5' exonuclease activity, and it is capable of cutting the ends of DNA for repair. Therefore, when exposed to toxic agents, one may observe exonucleolytic degradation of nascent DNA, which is the shortening of the DNA strand due to exposure to a DNA-damaging agent³⁴.

Replication fork breakages brought on by physical obstructions (DNA-protein complexes or DNA lesions), chemical impediments, or mutations may stop replication and necessitate homologous recombination to restart it. This is known as impaired fork progression. Numerous in vitro and in vivo investigations have indicated that transcription may, on occasion, prevent replication fork advancement in this manner³⁵.

Initiations are replication origins that initiate and fire during the first or second pulse. Origins that fire during the first pulse and have replication forks that continue to be active have a green-red-green pattern (Figure 2B, lower). Origins that initiate during the second pulse have a green-only pattern (Figure 2B, upper) and are sometimes called newly initiated origins, so those origins can be differentiated from those that initiate during the first pulse. The comparison of the relative percentages of newly fired origins between two experimental conditions allows one to understand how a cell responds to a DNA-damaging agent or the presence or absence of a protein. Terminations are created when two replication forks from adjacent replicons merge, and they have a red-green-red pattern (Figure 2D)³⁰.

Based on the facts described above, DNA fiber analysis is currently considered a preferred method to study the variation in DNA replication dynamics caused by toxic agents such as nanomaterials. Researchers now have a good understanding and knowledge of the dynamics of genome-wide DNA replication in eukaryotes, both quantitatively and qualitatively, owing to the discovery of this technology³⁶. Based on the outcome variables, several methodologies can be adopted. Some examples of methods to study the variations in DNA damage induced by external agents/nanoparticles are shown in Figure 3. The overall goal of the DNA fiber analysis method described in this study is to determine how nanoparticles impact the replication process in vitro and how they differentially affect various tissues.

Protocol

1. Preparation of antibodies and buffer

1. Prepare the primary antibody solution, mouse anti-BrdU at a 1:300 dilution in 5% BSA and rat anti-BrdU at a 1:500 dilution in 5% BSA.
2. Prepare the secondary antibody solution, alexafluor 594 rabbit anti-mouse at a 1:300 dilution in 5% BSA and alexafluor 488 chicken anti-rat at a 1:500 dilution in 5% BSA.
3. Prepare the tertiary antibody solution, alexafluor 594 goat anti-rabbit at a 1:1,000 dilution in 5% BSA and alexafluor 488 goat anti-chicken at a 1:1,000 dilution in 5% BSA.
4. Prepare 10 mL of lysis buffer in ddH₂O with 2 mL of 1M Tris (pH 7.4), 1 mL of 0.5 M EDTA, and 0.5 mL of 10% SDS.

2. Preparation for the fiber assay

1. Day 1: Cell culture and nanomaterial treatment for the fiber assay
   1. Plate RAW 264.5 macrophage cells or the cells of interest, so that the cells are in the log phase of their growth on the day of the experiment. For RAW cells, add 5 x 10⁴ cells/well to 24-well plates for each condition. Maintain the cells in a 37 °C incubator with 5% CO₂ and 98% humidity. Table 1 describes the constituents of the media used. Allow the cells to grow to 75%-80% confluency^17,37.
   2. After the cells reach the level of 75%-80%confluency, remove the medium from the plates, take half of the medium and reserve it for the CldU pulse (CldU reserve), and add 5 µL IdU to the other half at a final concentration of 50 µM. Mix and add the IdU-containing medium back to the plates. Place the cells with IdU in the incubator for 20 min.
   3. Place the CldU reserve medium at 37 °C to keep it pre-warmed. Add CldU to this medium just before the CldU pulse.
   4. After 20 min, aspirate the IdU-medium mixture, and gently wash the cells with 500 µL of phosphate-buffered saline (PBS, pH 7.4) by gently rotating the plate. Discard the PBS.
   5. Treat the cells with various concentrations of nanoparticles in 500 µL of the medium, and incubate for another 30 min. For this study, use treated graphene oxide nanoparticles at a 25 µg/mL concentration.
   6. Aspirate the treatment-medium mixture, and gently wash the cells with 500 µL of PBS by gently rotating the plate. Discard the PBS.Add 5 µL of CldU (100 µM final
   7. concentration) to the CldU reserve medium, and cover the cells with the CldU reserve medium. Place cells in the incubator for the duration of the CldU pulse for 20 min.
   8. Wash the cells with PBS, scrape off or trypsinize for 3-4 min, and then add 5 mL of medium to the plate, and collect the cells.
   9. Spin at 264 x g for 4 min. Remove the medium, and resuspend the cells in 1 mL of ice-cold PBS. Determine the cell numbers using a trypan blue assay (hemocytometer counting), and then dilute the cells to ~200-400 cells/µL. Keep the cell suspension on ice during the cell counting.
      NOTE: If possible, limit the time the cells sit on the ice. This may help obtain a bead of cell solution along the top of the slide. If they are kept on ice, keep them on for less than 30 min.
2. Day 1: Preparation of the DNA fibers
   1. Using a pencil, label the slides (glass slide, 75 mm x 25 mm) with the experimental condition and date.
   2. Take 2 µL of cells in a pipette, hold the pipette at a ~45° angle, place the pipette about 1 cm below the slide label, and move the pipette horizontally to the slide label. As the pipette moves across the slide, release a little of the cell solution at a time. Make multiple lines of cells on each slide (critical step).
      NOTE: There should be a horizontal line of cell suspension across the slide. If the cell suspension beads up, then add 5-10 µL of cell medium to the container that has the cell suspension, as this may help the solution to adhere to the slide when being applied again.
   3. Let the solution with the cells evaporate until the solution looks sticky and tacky. At this point, some liquid is associated with the cells, but not much. This step takes from 8-20 min and varies depending on how much humidity is in the air.
      NOTE: Ensure that all the solution has not evaporated, since this makes it very hard to obtain straight and aligned DNA fibers as most, if not all, of the DNA will be stuck together and unable to separate.
   4. When the solution is tacky, overlay the cells with 15 µL of spreading (lysis) buffer per each line of cells, taking care not to let the pipette tip touch the slide. Wait for 10 min. Tilt the slide at a 25° angle, and place the label of the slide horizontally against the edge of a tube rack (the bottom part of the label should line up with the edge of the tube rack).
   5. Allow the DNA spreads to air dry for a minimum of 4 h from the time the last slides were made.
      NOTE: A period of 4 h to 12 h is good for drying. However, do not let them dry overnight. If allowed to dry overnight, there will be a lot of relaxed DNA but very few straight and aligned DNA fibers.
3. Day 1: Fixing the DNA fibers and freezing
   1. Once the slides have dried, fix the slides with methanol:acetic acid (3:1) by immersing the slides for 2 min. Perform this step under a hood, and let the slides dry overnight in an area with limited light exposure, or cover the slides with a tin foil tent.
4. Day 2
   1. Place the slides in a glass slide carrier. Place the slides at −20 °C for a minimum of 24 h. This step improves the resolution or crispness of the image.

3. Performing the DNA fiber assay

1. Denaturation of the DNA
   1. Prepare a humidified chamber using small pipette tip boxes with lids. Half-fill the containers with water, and place them in a 37 °C water bath for at least 1 h.
   2. Take the slides out from −20 °C, and defrost them for a few seconds. Place the slides carefully into a Coplin jar containing deionized (DI) water so that the coated surfaces do not touch the walls of the jar. Incubate the slide for 20 s, and then carefully pour out the water.
   3. Add 2.5 M HCL to the Coplin jar such that it covers all the slides. Wait for 80 min. This is a critical step in the protocol. A change in the time can change the outcome of the image.
   4. Wash the slides 1x with PBS plus Tween (PBS + 0.1% Tween [final concentration]) and then 2x with PBS for 3 min each at room temperature (RT).
2. Immunostaining
   1. Keep the slides in the humidified chamber for the following steps. Block the slides in 5% BSA in PBS for 30 min at RT, and cover them with coverslips, coverslips made from a Western blot bag, or transparent plastic sheets.
   2. After blocking, carefully remove the coverslip, remove the blocking solution, and blot the slide on a paper towel (tap the slide on the towel) to remove the excess blocking solution.
   3. Add 100 µL of the primary antibody solution along the length of the slide. Add a new plastic coverslip, and wait for 2 h. When adding the coverslip, ensure that no bubbles form.
   4. After 2 h, knock off the antibody solution, and rinse the slides in a PBS-filled Coplin jar 2x at RT. Use fresh PBS each time to wash the slides.
   5. Block the slides again by adding 200 µL (three to four drops) of 5% BSA along each slide, and add a plastic coverslip. Wait for 15 min.
   6. Take off the coverslip, knock off the excess BSA on a paper towel, and add 100 µL of the secondary antibody solution along the length of the slide. Add a new plastic coverslip, and wait for 1 h.
   7. Take off the coverslip, knock off the excess BSA on a paper towel, and wash the slides 2x in PBS at RT.
   8. Block the slides again by adding 200 µL (three to four drops) of 5% BSA along each slide, and add a plastic coverslip. Wait for 15 min.
   9. If needed, remove the coverslip, remove the excess BSA on a paper towel, and add 100 µL of tertiary antibody solution along the length of the slide. Add new plastic coverslips, and wait for 30 min.
   10. Wash the slides with PBS 3x. Remove the excess PBS, and allow them to dry off completely.
   11. Once dry, add the mounting medium, and carefully place glass coverslips (24 mm x 60 mm) over the mounting medium so that bubbles are avoided. Re-label the slides, and leave them in the dark overnight.

4. Image acquisition

1. Visualize the immunostained DNA fibers using an immunofluorescent microscope equipped with a camera, a 60x objective, and appropriate filter sets to detect alexafluor 488 and 594 dyes.
2. Take images for the analysis in regions where the fibers are well separated and not entangled. It is important to capture images in different areas along the slide, as taking images in only one part of the slide may not provide representative data regarding all the replication intermediates found on the slide.
3. If possible, obtain fiber images from 20 different regions on a slide. Use only one channel to select the areas for taking images to avoid bias.

Results

After obtaining enough images (from 20-100 images per condition), the replication intermediates need to be identified, measured, and counted. Whether analyzing the fibers manually or automatically via a program³⁸, it is necessary to clearly define what characteristics a fiber must have for it to be counted or scored (or not counted or measured)³⁹. For instance, the following questions can be considered. (1) Should one measure and count only fibers with 100% immunof...

Discussion

We discuss here a method to assist in the analysis of the variation in DNA replication dynamics in the presence of nanoparticles through the DNA fiber assay. Major critical steps involved in the standard assay are described in the protocol (step 2.2.2 and step 3.1.3). It is always recommended to use an area with limited overhead light exposure and constant airflow to prevent light-induced DNA breaks in the slides as well as to enhance reproducibility. Careful attention is required on the time duration for the drying of t...

Materials

Name	Company	Catalog Number	Comments
24 well plate	Fisher brand	FB012929
Acetic Acid	Sigma Aldrich	695092
Alexa flour 594 goat anti-rabbit	Invitrogen	A11037
Alexa fluor 488 chicken anti-rat	Invitrogen	A21470
Alexa fluor 488 goat anti-chicken	Invitrogen	A11039
Alexa fluor 594 rabbit anti-mouse	Invitrogen	A11062
BSA	Sigma Aldrich	A2153
CldU	Sigma Aldrich	50-90-8
Coverslips (22 x 50 mm)	Fisher brand	12-545-EP
EDTA	Fisher Scientific	15575020
Frosted Microscope Slides	Fisher brand	12-550-11
Hydrochloric Acid	Sigma Aldrich	320331
IdU	Sigma Aldrich	54-42-2
Methanol	Fisher Scientific	A454-4
Mouse Anti-BrdU	BD Biosciences	347580
Phosphate Buffer Saline	Gibco	10010072
Rat anti-BrdU	Abcam	BU1--75(ICR1)
Raw 264.5 macrophage cells	ATCC	TIB-71
SDS	Sigma Aldrich	L3771
Silane-Prep slides	Sigma Aldrich	S4651-72EA
Superfrost gold plus slides	Fischer scientific	22-035813
Tris pH 7.4	Sigma Aldrich	77861
Tween 20	Sigma Aldrich	P9416