We thank the Dungrawala lab members for their input and feedback. This work was funded by NIH grant R35GM137800 to HD.

This protocol (Figure 1) primarily utilizes adherent U2OS cancer cell lines but is adaptable to a wide variety of adherent and suspension cells grown in tissue culture. The solutions and buffers used in this study are detailed in Table 1. The reagents and equipment used are listed in the Table of Materials.
1. Preparation of materials
<ol>
	<li>Prior to the day of the experiment, place the provided bottle with low melting ag...

Investigating the Extent of DNA Breaks Occurring During DNA Replication 

<ol>
	<li>Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+as+a+requirement+for+development+of+the+contact+effect+in+Chinese+hamster+v79+spheroids.">Cell proliferation as a requirement for development of the contact effect in Chinese hamster v79 spheroids.</a> Radiat Res. 117 (1), 79-92 (1989).</li><li>Olive, P. L., Banath, J. P., Durand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+radiation-induced+DNA+damage+and+repair+in+tumor+and+normal+cells+measured+using+the+&amp;#34;comet&amp;#34;+assay.">Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the &amp;#34;comet&amp;#34; assay.</a> Radiat Res. 122 (1), 86-94 (1990).</li><li>Singh, N. P., Mccoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells.">A simple technique for quantitation of low levels of DNA damage in individual cells.</a> Exp Cell Res. 175 (1), 184-191 (1988).</li><li>Olive, P. L., Banath, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+A+method+to+measure+DNA+damage+in+individual+cells.">The comet assay: A method to measure DNA damage in individual cells.</a> Nat Protoc. 1 (1), 23-29 (2006).</li><li>De Lapuente, J., 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=The+comet+assay+and+its+applications+in+the+field+of+ecotoxicology:+A+mature+tool+that+continues+to+expand+its+perspectives.">The comet assay and its applications in the field of ecotoxicology: A mature tool that continues to expand its perspectives.</a> Front Genet. 6, 180 (2015).</li><li>Azqueta, 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=Application+of+the+comet+assay+in+human+biomonitoring:+An+hcomet+perspective.">Application of the comet assay in human biomonitoring: An hcomet perspective.</a> Mutat Res Rev Mutat Res. 783, 108288 (2020).</li><li>Speit, G., Hartmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+A+sensitive+genotoxicity+test+for+the+detection+of+DNA+damage.">The comet assay: A sensitive genotoxicity test for the detection of DNA damage.</a> Methods Mol Biol. 291, 85-95 (2005).</li><li>Collins, 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=Measuring+DNA+modifications+with+the+comet+assay:+A+compendium+of+protocols.">Measuring DNA modifications with the comet assay: A compendium of protocols.</a> Nat Protoc. 18 (3), 929-989 (2023).</li><li>Rapp, A., Hausmann, M., Greulich, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet-fish+technique:+A+tool+for+detection+of+specific+DNA+damage+and+repair.">The comet-fish technique: A tool for detection of specific DNA damage and repair.</a> Methods Mol Biol. 291, 107-119 (2005).</li><li>Saldivar, J. C., Cortez, D., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+kinase+atr:+Ensuring+faithful+duplication+of+a+challenging+genome.">The essential kinase atr: Ensuring faithful duplication of a challenging genome.</a> Nat Rev Mol Cell Biol. 18 (10), 622-636 (2017).</li><li>Cimprich, K. A., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atr:+An+essential+regulator+of+genome+integrity.">Atr: An essential regulator of genome integrity.</a> Nat Rev Mol Cell Biol. 9 (8), 616-627 (2008).</li><li>Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-coupled+DNA+repair.">Replication-coupled DNA repair.</a> Mol Cell. 74 (5), 866-876 (2019).</li><li>Strumberg, 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=Conversion+of+topoisomerase+I+cleavage+complexes+on+the+leading+strand+of+ribosomal+DNA+into+5'-phosphorylated+DNA+double-strand+breaks+by+replication+runoff.">Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff.</a> Mol Cell Biol. 20 (11), 3977-3987 (2000).</li><li>Pommier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+I+inhibitors:+Camptothecins+and+beyond.">Topoisomerase I inhibitors: Camptothecins and beyond.</a> Nat Rev Cancer. 6 (10), 789-802 (2006).</li><li>Hsiang, Y. H., Hertzberg, R., Hecht, S., Liu, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Camptothecin+induces+protein-linked+DNA+breaks+via+mammalian+DNA+topoisomerase+i.">Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase i.</a> J Biol Chem. 260 (27), 14873-14878 (1985).</li><li>Bryant, H. E., 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=Specific+killing+of+brca2-deficient+tumours+with+inhibitors+of+poly(adp-ribose)+polymerase.">Specific killing of brca2-deficient tumours with inhibitors of poly(adp-ribose) polymerase.</a> Nature. 434 (7035), 913-917 (2005).</li><li>Helleday, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+underlying+mechanism+for+the+parp+and+BRCA+synthetic+lethality:+Clearing+up+the+misunderstandings.">The underlying mechanism for the parp and BRCA synthetic lethality: Clearing up the misunderstandings.</a> Mol Oncol. 5 (4), 387-393 (2011).</li><li>Mcglynn, A. P., 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=The+bromodeoxyuridine+comet+assay:+Detection+of+maturation+of+recently+replicated+DNA+in+individual+cells.">The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells.</a> Cancer Res. 59 (23), 5912-5916 (1999).</li><li>Morocz, M., Gali, H., Rasko, I., Downes, C. S., Haracska, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+human+rad18-dependent+DNA+post-replication+repair+by+alkaline+bromodeoxyuridine+comet+assay.">Single cell analysis of human rad18-dependent DNA post-replication repair by alkaline bromodeoxyuridine comet assay.</a> PLoS One. 8 (8), e70391 (2013).</li><li>Thakar, T., 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=Lagging+strand+gap+suppression+connects+BRCA-mediated+fork+protection+to+nucleosome+assembly+through+PCNA-dependent+CAF-1+recycling.">Lagging strand gap suppression connects BRCA-mediated fork protection to nucleosome assembly through PCNA-dependent CAF-1 recycling.</a> Nat Commun. 13 (1), 5323 (2022).</li><li>Vaitsiankova, 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=Parp+inhibition+impedes+the+maturation+of+nascent+DNA+strands+during+DNA+replication.">Parp inhibition impedes the maturation of nascent DNA strands during DNA replication.</a> Nat Struct Mol Biol. 29 (4), 329-338 (2022).</li><li>Dungrawala, 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=Radx+promotes+genome+stability+and+modulates+chemosensitivity+by+regulating+rad51+at+replication+forks.">Radx promotes genome stability and modulates chemosensitivity by regulating rad51 at replication forks.</a> Mol Cell. 67 (3), 374-386.e5 (2017).</li><li>Townsend, A., Lora, G., Engel, J., Tirado-Class, N., Dungrawala, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dcaf14+promotes+stalled+fork+stability+to+maintain+genome+integrity.">Dcaf14 promotes stalled fork stability to maintain genome integrity.</a> Cell Rep. 34 (4), 108669 (2021).</li><li>Lemacon, 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> Nat Commun. 8 (1), 860 (2017).</li><li>Leung, W., 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=Atr+protects+ongoing+and+newly+assembled+DNA+replication+forks+through+distinct+mechanisms.">Atr protects ongoing and newly assembled DNA replication forks through distinct mechanisms.</a> Cell Rep. 42 (7), 112792 (2023).</li><li>Bhowmick, R., Mehta, K. P. M., Lerdrup, M., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrator+facilitates+RNApii+removal+to+prevent+transcription-replication+collisions+and+genome+instability.">Integrator facilitates RNApii removal to prevent transcription-replication collisions and genome instability.</a> Mol Cell. 83 (13), 2357-2366.e8 (2023).</li><li>Mosler, T., 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=R-loop+proximity+proteomics+identifies+a+role+of+ddx41+in+transcription-associated+genomic+instability.">R-loop proximity proteomics identifies a role of ddx41 in transcription-associated genomic instability.</a> Nat Commun. 12 (1), 7314 (2021).</li><li>Petermann, E., Orta, M. L., Issaeva, N., Schultz, N., Helleday, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea-stalled+replication+forks+become+progressively+inactivated+and+require+two+different+rad51-mediated+pathways+for+restart+and+repair.">Hydroxyurea-stalled replication forks become progressively inactivated and require two different rad51-mediated pathways for restart and repair.</a> Mol Cell. 37 (4), 492-502 (2010).</li><li>Sestili, P., Cantoni, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotically+driven+radial+diffusion+of+single-stranded+DNA+fragments+on+an+agarose+bed+as+a+convenient+measure+of+DNA+strand+scission.">Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission.</a> Free Radic Biol Med. 26 (7-8), 1019-1026 (1999).</li><li>Sestili, P., Martinelli, C., Stocchi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fast+halo+assay:+An+improved+method+to+quantify+genomic+DNA+strand+breakage+at+the+single-cell+level.">The fast halo assay: An improved method to quantify genomic DNA strand breakage at the single-cell level.</a> Mutat Res. 607 (2), 205-214 (2006).</li><li>Gorczyca, W., Gong, J., Darzynkiewicz, Z. <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+DNA+strand+breaks+in+individual+apoptotic+cells+by+the+in+situ+terminal+deoxynucleotidyl+transferase+and+nick+translation+assays.">Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.</a> Cancer Res. 53 (8), 1945-1951 (1993).</li><li>Hanada, 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=The+structure-specific+endonuclease+mus81+contributes+to+replication+restart+by+generating+double-strand+DNA+breaks.">The structure-specific endonuclease mus81 contributes to replication restart by generating double-strand DNA breaks.</a> Nat Struct Mol Biol. 14 (11), 1096-1104 (2007).</li></ol>

The authors declare no competing interests.

This protocol outlines a NC assay for assessing DNA breaks in human cells undergoing active replication. The protocol is relatively simple to perform, and researchers can readily adapt it to high pH conditions for alkaline analyses8. It includes two critical steps, as described in steps 3.7 and 4.5. In step 3.7, it is essential to spread the melted agarose with cells uniformly across the well to prevent comets from overlapping during analysis. Ensure that cellular aggregates are dissolved before r...

The comet assay is a gel electrophoresis method used to detect DNA breaks at the single-cell level. It relies on the principle that open-ended DNA breaks migrate under electrophoretic conditions, while intact DNA remains largely static. Broken DNA migrates because breaks result in the relaxation of supercoiled DNA, causing its gradual spatial relocation towards the anode in the electrophoretic chamber, which results in a comet-like appearance observed by immunofluorescence1,2. The extent of DNA damage is then measured by quantifying the amount of broken DNA that has migrated relative to the compact DNA.
The two most commonly used comet assay methods are the alkaline comet (AC) assay and the neutral comet (NC) assay. The AC assay is performed under denaturing conditions using a high alkaline pH solution, while the NC assay is carried out in a solution with neutral pH. Both NC and AC assays can reliably detect DNA breaks that occur in the nucleus. However, the alkaline version is also advantageous for detecting alkali-labile sites3. Both formats of the assay require the use of lysis conditions to ensure that DNA is free of proteins prior to electrophoresis.
This assay is a simple method for detecting DNA breaks and offers several unique advantages for determining cellular DNA damage. The method is relatively easy to set up and requires reagents that can either be prepared in the laboratory or purchased from commercial vendors. As a single-cell resolution technique, the assay requires very little starting material. Notably, the NC assay can measure breaks with high sensitivity, reported to detect between 50 and 10,000 breaks per cell4. The versatility of this technique is demonstrated by its wide range of applications, including ecotoxicology5, human biomonitoring6, and genotoxicity studies7. The assay can also be reliably used across various cell types and adapted for high-throughput assays8 and for assessing breaks at specific genomic regions9. Thus, this assay serves as a rapid and reliable technique for investigating break formation at the single-cell level.
The process of DNA replication is constantly challenged by several endogenous and exogenous stressors10,11. Stalling of replisomes due to such lesions can cause DNA breaks and result in heightened genome instability. DNA breaks also often arise as replication intermediates to facilitate DNA repair12. Additionally, several chemotherapeutic agents induce replication-dependent double-strand breaks (DSBs), such as camptothecin (CPT)13,14,15and PARP inhibitors16,17. Thus, this assay serves as a powerful methodology to study the nature of DNA lesions, assess the processing of replication forks, and investigate the therapeutic potential of DNA-damaging agents. Adaptations of the assay that involve labeling newly synthesized DNA with BrdU have been useful for studying replication-associated stress phenotypes18,19,20,21. Notably, the NC assay is a reliable method for studying DNA break induction in the context of replication, as demonstrated in studies involving the characterization of fork proteins22,23, analysis of replication intermediates24,25, and investigation of transcription-replication conflicts in genome maintenance26,27. Herein, we outline a NC assay protocol with the goal of quantifying DNA breaks during replication in human cells. This protocol can be readily implemented by researchers interested in assessing replication stress-associated damage in a wide variety of dividing human cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x DPBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Camptothecin</td>
			<td>Selleckchem</td>
			<td>S1288</td>
			<td></td>
		</tr>
		<tr>
			<td>Comet LMAgarose (LMA)</td>
			<td>R&#38;D systems</td>
			<td>4250-050-02</td>
			<td></td>
		</tr>
		<tr>
			<td>CometAssay Electrophoresis System II</td>
			<td>R&#38;D systems</td>
			<td>4250-050-ES</td>
			<td>Includes electrophoresis tank, safety lid, cables, 2/20 wells slide trays and slide tray overlay</td>
		</tr>
		<tr>
			<td>CometScore software</td>
			<td>TriTek</td>
			<td></td>
			<td>open-sourced</td>
		</tr>
		<tr>
			<td>CometSlide</td>
			<td>R&#38;D systems</td>
			<td>4250-050-03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>FisherSci</td>
			<td>D128-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Keyence</td>
			<td>BZ-X810</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium</td>
			<td>Bio-Techne</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 10.0</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>hTERT-RPE-1 cells</td>
			<td>ATCC</td>
			<td>CRL-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyurea</td>
			<td>Millipore Sigma</td>
			<td>H8627</td>
			<td></td>
		</tr>
		<tr>
			<td>PARP inhibitor Olaparib</td>
			<td>Selleckchem</td>
			<td>S8096</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac&#160;</td>
			<td>FisherSci</td>
			<td>FB300Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Surface Treated Sterile Tissue Culture Plate</td>
			<td>FisherSci</td>
			<td>FB012927</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green I Nucleic Acid Gel Stain (10,000x)</td>
			<td>FisherSci</td>
			<td>S7567</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP&#160;HB-1000 Hybridization Incubator</td>
			<td>FisherSci</td>
			<td>UVP95003001</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of dna breaks in dividing human cells by neutral comet assay

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.

Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

The main objective of our lab is to study mechanisms that promote genome duplication in human cells. Our project focuses on understanding how chromatin remodelers act at replication forks to promote DNA synthesis. Forks undergo multiple processing events when exposed to replication stress.These events give rise to replication intermediates that can convert to toxic breaks if left unrepaired. Recent studies demonstrate that the presence of single-strand DNA gaps in nascent DNA can cause genome instability and is a determinant of chemo sensitivity. Our work addresses the role of a conserved nucleosome remodeler, SNF2L, in DNA synthesis.SNF2L travels with replication forks, but the mechanism of action is poorly characterized. Using the comet assay protocol allows us to assess consequences in fork stability when SNF2L is absent.

Analysis of break induction by replication stress reagents 
U2OS cells were treated with DMSO, 4 of mM hydroxyurea (HU), or 100 nM of CPT for 4 h and analyzed for break accumulation by the NC assay (Figure 6A). While CPT induces breaks during S-phase by blocking topoisomerase-113, HU-induced depletion of nucleotide pools stalls replication forks that are progressively converted into DSBs28. The data indicates that exposure...

This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during ...

detection-of-dna-breaks-in-dividing-human-cells-by-neutral-comet-assay

Research

JoVE Journal

Bioengineering

Analyzing DNA Break Induction in U2OS Cancer Cells Using Comet Assay

To begin, remove the 1.5 milliliter centrifuge tubes containing 100 microliters of 1%solidified argos from the four degrees Celsius storage. Place the tubes in a water bath at 42 degrees Celsius to melt the agros. Mark the sample identifications on the frosted end of the two well comet slides.Seed adherent U2 OS cancer cells in a six well dish containing DMEM and culture overnight at 37 degrees Celsius with 5%carbon dioxide. The next day to demonstrate replication dependent break induction treat cells with one micromolar Camptothecin or CPT for one hour. After treatment aspirate the medium and rinse the cells twice with PBS, add 300 microliters of trypsin to each well.Incubate the dish in a tissue culture incubator for two to three minutes. Now add 700 microliters of DMEM to each well. Resuspend gently and transfer the suspension to 1.5 milliliter centrifuge tubes.Centrifuge the cell suspension at 2, 400 G for five minutes. Remove the supernatant and resuspend the pellet in one milliliter of cold PBS. After the final centrifugation, resuspend the cells in one milliliter of PBS at a cell density of two times 10 to the power of five cells per milliliter.Transfer 10 microliters of the cell suspension to a 100 microliter aliquot of melted argos. Using the same pipette tip mixed gently and spread the mixture evenly into one well of the slide. Store the slides at four degrees Celsius in the dark for 15 to 20 minutes.After solidification, transfer the slides to coplan jars containing lysis buffer for one hour, rinse the slides once with a cold TAE buffer. Then immerse the slides in a cold TAE buffer for 30 minutes. Pour approximately 850 milliliters of cold TAE buffer into the electrophoresis apparatus.Place the slides on the electrophoresis tray in the proper orientation to allow for the migration of broken DNA. Gently place the slide tray overlay on top of the tray to cover the slides. Connect the cables and set the power to 21 volts.Perform electrophoresis for 40 minutes. After electrophoresis remove the slides and immerse them in a DNA precipitation solution for 30 minutes. Transfer the slides to a 70%ethanol solution for 30 minutes.Dry the slides in a hybridization oven at 45 degrees Celsius for 30 minutes in the dark. Next, add 100 microliters of cyber green solution onto the slides and incubate in the dark. After 30 minutes, tilt the slide and remove the excess dye solution by gently dabbing the edge of the well with a lint-free wipe.Dry the slides in a drawer for 30 minutes. After acquiring images for at least 100 comets per sample, open the COMET score software and export images score. The comets that are individually distinguishable and fully contained in the field of view.Break induction significantly increased in U2 OS cells treated with CPT and hydroxyurea or HU compared to DMSO treated controls. Lysis temperature had a minimal effect on tail migration in untreated U2 OS cells. Prolonged HU treatment in U2 OS cells led to an increase in break induction over time.Increased CPT concentration resulted in a dose-dependent increase in break accumulation in U2 OS cells. Basal DNA break levels varied among hTERT-RPE-1, HEK293T, and U2 OS cells with increased breaks observed following CPT treatment. Break accumulation was observed in U2 OS cells treated with HU CPT and PARP inhibitor as compared to DMSO controls.

227 Views.  University of South Florida, Tampa.  This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.

Investigating the Extent of DNA Breaks Occurring During DNA Replication

Summary