Name: demonstration of the dna fiber assay for investigating dna damage and repair dynamics induced by nanoparticles
Uploaded: 2023-03-03T13:43:00
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...

<ol>
	<li>Waga, S., Stillman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+replication+fork+in+eukaryotic+cells.">The DNA replication fork in eukaryotic cells.</a> Annual Review of Biochemistry. 67 (1), 721-751 (1998).</li><li>Gambus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Termination+of+eukaryotic+replication+forks.">Termination of eukaryotic replication forks.</a> DNA Replication. 1042, 163-187 (2017).</li><li>Berti, M., Cortez, D., Lopes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+DNA+replication+forks+in+response+to+clinically+relevant+genotoxic+stress.">The plasticity of DNA replication forks in response to clinically relevant genotoxic stress.</a> Nature Reviews Molecular Cell Biology. 21 (10), 633-651 (2020).</li><li>Carr, A. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioresistant+DNA+synthesis:+An+intrinsic+feature+of+ataxia+telangiectasia.">Radioresistant DNA synthesis: An intrinsic feature of ataxia telangiectasia.</a> Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 84 (1), 183-190 (1981).</li><li>Aten, J. A., Bakker, P. J. M., Stap, J., Boschman, G. A., Veenhof, C. H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double+labelling+with+IdUrd+and+CldUrd+for+spatial+and+temporal+analysis+of+cell+proliferation+and+DNA+replication.">DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication.</a> The Histochemical Journal. 24 (5), 251-259 (1992).</li><li>Gratzner, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+to+5-bromo-and+5-iododeoxyuridine:+A+new+reagent+for+detection+of+DNA+replication.">Monoclonal antibody to 5-bromo-and 5-iododeoxyuridine: A new reagent for detection of DNA replication.</a> Science. 218 (4571), 474-475 (1982).</li><li>Huberman, J. A., Riggs, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiography+of+chromosomal+DNA+fibers+from+Chinese+hamster+cells.">Autoradiography of chromosomal DNA fibers from Chinese hamster cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 55 (3), 599-606 (1966).</li><li>Ockey, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+replicon+behavior+between+X-irradiation-sensitive+L5178Y+mouse+lymphoma+cells+and+AT+fibroblasts+using+DNA+fiber+autoradiography.">Differences in replicon behavior between X-irradiation-sensitive L5178Y mouse lymphoma cells and AT fibroblasts using DNA fiber autoradiography.</a> Radiation Research. 94 (2), 427-438 (1983).</li><li>Brayner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+toxicological+impact+of+nanoparticles.">The toxicological impact of nanoparticles.</a> Nano Today. 3 (1-2), 48-55 (2008).</li><li>Mendez-Bermudez, A., 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=Genome-wide+control+of+heterochromatin+replication+by+the+telomere+capping+protein+TRF2.">Genome-wide control of heterochromatin replication by the telomere capping protein TRF2.</a> Molecular Cell. 70 (3), 449-461 (2018).</li><li>Vindigni, A., Lopes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+electron+microscopy+with+single+molecule+DNA+fiber+approaches+to+study+DNA+replication+dynamics.">Combining electron microscopy with single molecule DNA fiber approaches to study DNA replication dynamics.</a> Biophysical Chemistry. 225, 3-9 (2017).</li><li>Herrick, J., Bensimon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+analysis+of+DNA+replication.">Single molecule analysis of DNA replication.</a> Biochimie. 81 (8-9), 859-871 (1999).</li><li>Taylor, J. H., Miner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Units+of+DNA+replication+in+mammalian+chromosomes.">Units of DNA replication in mammalian chromosomes.</a> Cancer Research. 28 (9), 1810-1814 (1968).</li><li>Nieminuszczy, J., Schwab, R. A., Niedzwiedz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+fibre+technique-tracking+helicases+at+work.">The DNA fibre technique-tracking helicases at work.</a> Methods. 108, 92-98 (2016).</li><li>Lema&#231;on, D., 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=MRE11+and+EXO1+nucleases+degrade+reversed+forks+and+elicit+MUS81-dependent+fork+rescue+in+BRCA2-deficient+cells.">MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells.</a> Nature Communications. 8, 860 (2017).</li><li>Vindigni Lab. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+Fork+Protection.">Replication Fork Protection.</a> Washington University School of Medicine. , (2020).</li><li>Mimitou, E. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+resection:+Many+nucleases+make+light+work.">DNA end resection: Many nucleases make light work.</a> DNA Repair. 8 (9), 983-995 (2009).</li><li>Schlacher, K., 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=Double-strand+break+repair-independent+role+for+BRCA2+in+blocking+stalled+replication+fork+degradation+by+MRE11.">Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.</a> Cell. 145 (4), 529-542 (2011).</li><li>Prado, F., Aguilera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+replication+fork+progression+mediates+RNA+polII+transcription-associated+recombination.">Impairment of replication fork progression mediates RNA polII transcription-associated recombination.</a> The EMBO Journal. 24 (6), 1267-1276 (2005).</li><li>Jackson, D. A., Pombo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicon+clusters+are+stable+units+of+chromosome+structure:+Evidence+that+nuclear+organization+contributes+to+the+efficient+activation+and+propagation+of+S+phase+in+human+cells.">Replicon clusters are stable units of chromosome structure: Evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells.</a> The Journal of Cell Biology. 140 (6), 1285-1295 (1998).</li><li>Halliwell, J. A., Gravells, P., Bryant, H. 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. 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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.

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	<tbody>
		<tr>
			<td>24 well plate</td>
			<td>Fisher brand</td>
			<td>FB012929</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid&#160;</td>
			<td>Sigma Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa flour 594 goat anti-rabbit&#160;</td>
			<td>Invitrogen</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 chicken anti-rat&#160;&#160;</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488 goat anti-chicken&#160;</td>
			<td>Invitrogen</td>
			<td>A11039</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 594 rabbit anti-mouse&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Abcam</td>
			<td>BU1--75(ICR1)</td>
			<td></td>
		</tr>
		<tr>
			<td>Raw 264.5 macrophage cells&#160;</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&#160;</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 ...

DNA fiber analysis allows us to understand how nanomaterials impact DNA at single-molecular level. And this information will enable developing strategies that will reduce nanotoxicity. This technique allows us to understand DNA dynamics and how it's impacted when cells or tissues are exposed to DNA damaging agents, either exogenous or endogenous, and we can look at DNA replication, DNA repair, and chromatin dynamics Studying DNA replication dynamics and repair mechanism will assist in developing strategies that will improve the efficacy of therapeutic material or reduce the off-target effect of nanomedicine.What we really want people to know about this technique is it's the different things that we can or one person can do. One is to understand how novel chemotherapeutic agents can impact the health of a cell, to see if you can kill them faster or more efficiently. The next thing is to think about when you are adding an agent, let's say it's a new therapeutic or pharmaceutical agent, you wanna know if it's gonna hurt a person before you give it to them.So therefore, you can just give the agent to cells or model systems and see if it hurts the cells or the model system, and if so, how much? The next thing is to kinda look at, we're developing all these new nanomedicines and we wanna make sure that those nanomedicines doesn't harm a person or the place where we're injecting the nanomedicine. The last thing is sometimes you wanna know how a protein impacts DNA replication, DNA repair, or chromatin dynamics and we can use this technique to be able to do that.This is Shirali Patel, a master's in medical biotechnology research student in my laboratory, will be demonstrating the procedure. Begin the fiber assay preparation by removing the medium from 75%to 80%confluent RAW 264.5 macrophage cells and dividing it into two halves. Reserve one half for the chloro-iododeoxyuridine pulse or chloro-iododeoxyuridine reserve and add five microliters of iododeoxyuridine to the other half, making 50 micromoles as the final concentration.After mixing, add the iododeoxyuridine-containing medium back to the plates and place the cells in the incubator for 20 minutes. Pre-warm the chloro-iododeoxyuridine reserve medium at 37 degrees Celsius and add chloro-iododeoxyuridine to this medium before the chloro-iododeoxyuridine pulse. After 20 minutes, aspirate the iododeoxyuridine medium mixture from the RAW 264.5 macrophage cell flask.Gently wash the cells with 500 microliters of PBS, having 7.4 pH, and rotate the plate before discarding the PBS. Treat the cells with various concentrations of graphene oxide nanoparticles in 500 microliters of the medium and incubate for 30 minutes. After incubation, aspirate the treatment-medium mixture and gently wash the cells with 500 microliters of PBS by gently rotating the plate before discarding the PBS.Next, add five microliters of chloro-iododeoxyuridine to the chloro-iododeoxyuridine reserve medium, and cover the cells with the chloro-iododeoxyuridine reserve medium. For the chloro-iododeoxyuridine pulse, place the cells in the incubator for 20 minutes. After a PBS wash, scrape off the cells for three to four minutes, then add five milliliters of medium to the plate and collect the cells.Centrifuge the collected cells at 264G for four minutes. Remove the supernatant and re-suspend the cells in one milliliter of ice-cold PBS. To prepare the DNA fibers, label the glass slides with the experimental condition and date, using a pencil.Aspirate two microliters of cells in a pipette and hold the pipette at an approximately 45 degree angle. Then place the pipette about one centimeter below the slide label and move the pipette horizontally towards the slide label with a slow release of cell solution. Once the solution evaporates and looks sticky and tacky, overlay each line of cells with 15 microliters of lysis buffer without letting the pipette tip touch the slide.Then tilt the slide at a 25 degree angle and allow the DNA spreads to air dry for a minimum of four hours. On day one, fix the DNA fibers by immersing the slides in methanol acetic acid for two minutes. On day two, place the slides in a glass slide carrier and incubate them at 20 degrees Celsius for a minimum of 24 hours.To denature the DNA, prepare a humidified chamber and place the slides carefully into a Coplin jar containing deionized, or DI, water, ensuring the coated surfaces do not touch the walls of the jar. After 20 seconds of incubation, pour out the water. Add 2.5 molar hydrochloric acid to the Coplin jar, covering all the slides.After 80 minutes of incubation, give one wash with PBS plus 0.1%Tween, followed by two washes with PBS for three minutes each at room temperature. For immunostaining, place the slides in the humidified chamber and block them using 5%bovine serum albumin, or BSA, in PBS for 30 minutes at room temperature. Then cover the slides with a transparent plastic sheet.After blocking, remove the cover slip and add 100 microliters of the primary antibody solution along the slide length. Add a new plastic cover slip and wait for two hours. After two hours, knock off the antibody solution and rinse the slides in a PBS-filled Coplin jar two times.Block the slides again by adding 200 microliters of 5%BSA along the slide. Add a plastic cover slip and incubate for 15 minutes. After taking off the cover slip and removing excess BSA using a paper towel, add 100 microliters of the secondary antibody solution along the slide length and place a new plastic cover slip.After one hour, remove the cover slip, knock off the excess BSA on a paper towel, and wash the slides two times in PBS. Add 100 microliters of the tertiary antibody solution along the slide length and place a new plastic cover slip. Again, add 200 microliters of 5%BSA along each slide and incubate for 15 minutes.After three washes with PBS, remove the excess PBS and allow them to dry off completely. Once dry, add the mounting medium on the slide and place glass cover slips without allowing bubble formation. Visualize the immunostained DNA fibers using an immunofluorescent microscope equipped with a camera, a 60x objective, and appropriate filter sets to detect Alexa Fluor 488 and 594 dyes.The variation in replication patterns after long-term exposure to the nanomaterials was determined by comparing the replication patterns of cells before and after the iododeoxyuridine and chloro-iododeoxyuridine nanoparticle exposure. DNA fiber analysis and interpretation images revealed a pattern for control macrophage cells and graphene-oxide-nanoparticle-treated macrophage DNA. The variation was observed in the pattern of replication intermediates, showing an increase in new origins on a different region of the same condition.The conclusive fiber data was obtained by dividing the total area of the slide into five-to-six regions and taking multiple images from each region. Selecting around 500 intermediates from multiple slides or conditions was ideal to investigate the variation in DNA replication dynamics. The analysis also indicated an increase in the red-only tracks, indicating determinations and a decrease in newly fired origins in the graphene-oxide-treated cells Compared to the control.The quality of the DNA fiber spread depends upon how well the fibers spread out from each other and that they do not overlap. Once the fibers have been spread out and IdU and CldU have been stained, it is extremely important to find the DNA fibers. One of the things you can do after you make the DNA fibers is, one, you can look at how replication dynamics is impacted.Two, you can look at where DNA damage is located relative to the DNA replication tracks. And the last thing is if you use the FISH probes, you can actually look at specific regions and how they're impacted by that DNA damage. This technique paves the way for development of chemotherapy drugs, drug design, and understanding how DNA replication is impacted by various types of proteins.

Research

JoVE Journal

Biology

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Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

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Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

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Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

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