Name: demonstration of the dna fiber assay for investigating dna damage and repair dynamics induced by nanoparticles
Uploaded: 2023-03-03T13:43:00.000+00:00
Duration: 13 min 9 s
Description: Watch this Scientific Journal Video about Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles at JoVE.com

The authors acknowledge financial support from the Blazer foundation, the Medical Biotechnology Program at Biomedical Sciences, UIC Rockford, and the Department of Health Science Education, UIC Rockford. The authors thank Ananya Sangineni and James Bradley for their contributions to the project.

1. Preparation of antibodies and buffer
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Preparation for the fiber assay
<ol>
	<li>Day 1: Cell culture and nanomaterial treatment for the fiber assay
	<ol>
		<li>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 &#176;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% confluency17,37.</li>
		<li>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 &#181;L IdU to the other half at a final concentration of 50 &#181;M. Mix and add the IdU-containing medium back to the plates. Place the cells with IdU in the incubator for 20 min.</li>
		<li>Place the CldU reserve medium at 37 &#176;C to keep it pre-warmed. Add CldU to this medium just before the CldU pulse.</li>
		<li>After 20 min, aspirate the IdU-medium mixture, and gently wash the cells with 500 &#181;L of phosphate-buffered saline (PBS, pH 7.4) by gently rotating the plate. Discard the PBS.</li>
		<li>Treat the cells with various concentrations of nanoparticles in 500 &#181;L of the medium, and incubate for another 30 min. For this study, use treated graphene oxide nanoparticles at a 25 &#181;g/mL concentration.</li>
		<li>Aspirate the treatment-medium mixture, and gently wash the cells with 500 &#181;L of PBS by gently rotating the plate. Discard the PBS.Add 5 &#181;L of CldU (100 &#181;M final</li>
		<li>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.</li>
		<li>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.</li>
		<li>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/&#181;L. 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.</li>
	</ol>
	</li>
	<li>Day 1: Preparation of the DNA fibers
	<ol>
		<li>Using a pencil, label the slides (glass slide, 75 mm x 25 mm) with the experimental condition and date.</li>
		<li>Take 2 &#181;L of cells in a pipette, hold the pipette at a ~45&#176; 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 &#181;L 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.</li>
		<li>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.</li>
		<li>When the solution is tacky, overlay the cells with 15 &#181;L 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&#176; 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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Day 1: Fixing the DNA fibers and freezing
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Place the slides in a glass slide carrier. Place the slides at &#8722;20 &#176;C for a minimum of 24 h. This step improves the resolution or crispness of the image.</li>
	</ol>
	</li>
</ol>
3. Performing the DNA fiber assay
<ol>
	<li>Denaturation of the DNA
	<ol>
		<li>Prepare a humidified chamber using small pipette tip boxes with lids.&#160;Half-fill the containers with water, and place them in a 37 &#176;C water bath for at least 1 h.</li>
		<li>Take the slides out from &#8722;20 &#176;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.</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Add 100 &#181;L 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.</li>
		<li>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.</li>
		<li>Block the slides again by adding 200 &#181;L (three to four drops) of 5% BSA along each slide, and add a plastic coverslip. Wait for 15 min.</li>
		<li>Take off the coverslip, knock off the excess BSA on a paper towel, and add 100 &#181;L of the secondary antibody solution along the length of the slide. Add a new plastic coverslip, and wait for 1 h.</li>
		<li>Take off the coverslip, knock off the excess BSA on a paper towel, and wash the slides 2x in PBS at RT.</li>
		<li>Block the slides again by adding 200 &#181;L (three to four drops) of 5% BSA along each slide, and add a plastic coverslip. Wait for 15 min.</li>
		<li>If needed, remove the coverslip, remove the excess BSA on a paper towel, and add 100 &#181;L of tertiary antibody solution along the length of the slide. Add new plastic coverslips, and wait for 30 min.</li>
		<li>Wash the slides with PBS 3x. Remove the excess PBS, and allow them to dry off completely.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Image acquisition
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+fiber+assay+for+the+analysis+of+DNA+replication+progression+in+human+pluripotent+stem+cells.">DNA fiber assay for the analysis of DNA replication progression in human pluripotent stem cells.</a> Current Protocols in Stem Cell Biology. 54 (1), 115 (2020).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+DNA+fiber+tracking+and+measurement.">Automated DNA fiber tracking and measurement.</a> 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro. , 1349-1352 (2011).</li><li>Quinet, A., Carvajal-Maldonado, D., Lemacon, D., Vindigni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+fiber+analysis:+Mind+the+gap.">DNA fiber analysis: Mind the gap.</a> Methods in Enzymology. 591, 55-82 (2017).</li><li>T&#233;cher, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+dynamics:+Biases+and+robustness+of+DNA+fiber+analysis.">Replication dynamics: Biases and robustness of DNA fiber analysis.</a> Journal of Molecular Biology. 425 (23), 4845-4855 (2013).</li><li>Martins, D. J., Tirman, S., Quinet, A., Menck, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+post-replicative+gaps+accumulation+and+repair+in+human+cells+using+the+DNA+fiber+assay.">Detection of post-replicative gaps accumulation and repair in human cells using the DNA fiber assay.</a> Journal of Visualized Experiments. (180), e63448 (2022).</li></ol>

The authors have no conflicts of interest to disclose.

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...

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.&#160;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 applications7,8. 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 region9,10.
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 used13,14,15,16.
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&#39;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 assessed17,18. 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 cells14,20. 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&#8242;-deoxyuridine) for flow cytometry to measure the overall rates of DNA synthesis21,22. 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 this23,24,25,26.
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 autoradiography27,28. 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&#39;-5&#39; 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.

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

demonstration of the dna fiber assay for investigating dna damage and repair dynamics induced by nanoparticles

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.

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...

Watch this Scientific Journal Video about Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles at JoVE.com

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

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.

Nanomaterial exposure can cause replication stress and genomic instability in cells. The degree of instability depends on the chemistry, size, and ...

demonstration-dna-fiber-assay-for-investigating-dna-damage-repair

Research

JoVE Journal

Biology

2.9K Views.  University of Illinois College of Medicine Rockford. 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.

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

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Genetics

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Cancer Research

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

Quantifying DNA Double Strand Break Repair with Nanoluciferase Reporter Assays

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Author Spotlight: Developing Novel Anticancer Therapeutics Targeting the DNA Damage Response

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

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

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Chapters in this video

Single Molecule Analysis of Laser Localized Psoralen Adducts

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Laser Micro-Irradiation to Study DNA Recruitment During S Phase