The work in C.F.M.M. laboratory is supported by Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP, S&#227;o Paulo, Brazil, Grants #2019/19435-3, #2013/08028-1 and 2017/05680-0) under the International Collaboration Research from FAPESP and The Netherlands Organization for Scientific Research (NWO, The Netherlands); Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq, Bras&#237;lia, DF, Brazil, Grants # 308868/2018-8] and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal do Ensino Superior (CAPES, Bras&#237;lia, DF, Brazil, Finance Code 001).

As the study uses human cells, the work was approved by the Ethics Committee of the Institute of Biomedical Science at the University of S&#227;o Paulo (ICB-USP, approval number #48347515.3.00005467) for the research with human samples.
NOTE: The protocols described here were used in previous publications with minor modifications14,16,28. Here the focus is on the use of ultraviolet light C (UVC) as a DNA damaging agent. However, other DNA damaging agents such as cisplatin and hydroxyurea have also been successfully employed28. This protocol can be performed in an array of cell lines, including U2OS, HEK293T, human fibroblasts, and others14,28,29. It is essential to determine the experimental question before performing this protocol. The DNA fiber assay with the S1 nuclease (called S1 Fiber hereafter) is used to detect the presence of post-replicative ssDNA gaps, while the gap-filling (or PRR) assay is performed to quantify gap-filling events in the G2 phase. Thus, if analyzing the presence of post-replicative ssDNA gaps, the investigator should perform section 2 (S1 Fiber experimental setup) and proceed directly to section 4 (DNA fiber preparation by spreading) of the protocol. If analyzing gap-filling events, the investigator should proceed directly to section 3 (Gap-filling (PRR) experimental setup).
1. Reagents and setup
<ol>
	<li>Thymidine analogs: Resuspend 5-Iodo-2&#39;-deoxyuridine (IdU) and 5-Chloro-2&#39;-deoxyuridine (CldU) in 1 N NH4OH to a final concentration of 100 mM. Filter the analogs through a sterile syringe filter (0.2 &#181;m), aliquot, and store them at -20 C.</li>
	<li>Antibodies: Primaries - anti-BrdU rat (Biorad) and anti-BrdU mouse (BD); Secondaries - anti-rat Alexa Fluor 488 and anti-mouse Alexa Fluor 594.</li>
	<li>Cellular permeabilization CSK100 buffer for the S1 Fiber: Add 100 mM NaCl, 10 mM MOPS [pH 7], 3 mM MgCl2 [pH 7.2], 300 mM sucrose and 0.5% Triton X-100 in distilled H2O.</li>
	<li>S1 nuclease buffer [pH 4.6]: Add 30 mM Sodium Acetate [pH 4.6], 10 mM Zinc Acetate, 50 mM NaCl and 5% Glycerol in H2O. 
	NOTE: The S1 nuclease buffer must have a final pH of 4.6, as the S1 activity drops 50% at pH &#62; 4.9.</li>
	<li>Aliquot and store at -20 &#176;C the S1 nuclease purified from A. oryzae pre-diluted in S1 nuclease dilution buffer provided by the manufacturer.</li>
	<li>Reconstitute the nocodazole in DMSO to a final concentration of 2 mM for gap-filling. assay.</li>
	<li>Lysis buffer: Add 200 mM Tris-HCl [pH 7.5], 50 mM EDTA and 0.5 % SDS in H2O.</li>
	<li>Prepare phosphate-buffered saline (PBS).</li>
	<li>Prepare PBS-T by adding 0.1% Tween-20 in PBS.</li>
	<li>Prepare 5% Bovine Serum Albumin (BSA) in PBS.</li>
	<li>Prepare 0.1% BSA in PBS.</li>
	<li>Prepare PBS-T-BSA by adding 1% BSA in PBS-T. 
	NOTE: Use an epifluorescent microscope with a mercury or xenon lamp with a 510 nm wavelength and 42 nm bandwidth GFP (FITC, Alexa Fluor 488) emission filter and a 624 nm wavelength and 40 nm bandwidth Texas Red (Alexa Fluor 594) emission filter or a confocal microscope with a 488-nm line (blue) laser to detect green fluorescence (detected between 510 and 560 nm) and a 561-nm line (yellow) laser to detect red fluorescence (detected between 575 and 680 nm). Immersion objective (40x, 63x or 100x).</li>
</ol>
2. S1 Fiber experimental setup (2 days)
<ol>
	<li>Day 1: Plate 4 wells for each cell line: &#177; UVC and &#177; S1 nuclease with cells at 70-90% confluency. 
	&#8203;NOTE: The experiment can be done in a format as small as a 12-well plate. In this case, all solutions are used at 0.5 mL/well.</li>
	<li>Day 2: Prepare fresh IdU at 20 &#181;M and CldU at 200 &#181;M in pre-warmed (37 &#176;C) cell culture media.</li>
	<li>Aspirate the culture media and immediately add the media with 20 &#181;M IdU and incubate the cells at 37 &#176;C, 5% CO2 (cell incubator) for precisely 20 min.</li>
	<li>Quickly wash the cells with pre-warmed (37 &#176;C) PBS twice.</li>
	<li>Irradiate the cells with 20 J/m2 UVC. Use untreated cells as controls.</li>
	<li>Immediately add the media with 200 &#181;M CldU and incubate the cells at 37 &#176;C, 5% CO2 (cell incubator) for precisely 60 min. 
	NOTE: IdU and CldU labeling can be interchanged, but the concentration of the second analog should be 5-10 times higher than the first analog. Timing of the analog incubation can be adapted to the genotoxic agent used, but the time of the second analog incubation should be long enough to ensure enough time for gap formation14,16. If using a DNA damaging drug, add the drug together with CldU28,29,30.</li>
	<li>Wash the cells with pre-warmed (37 &#176;C) PBS twice.</li>
	<li>Permeabilize the cells with the CSK100 buffer for 8-10 min at room temperature (RT). 
	NOTE: Successful permeabilization can be checked under a brightfield microscope where only nuclei should be observable</li>
	<li>Carefully wash the nuclei with PBS (pour 0.5 mL down the side of each well, just for a few seconds).</li>
	<li>Carefully wash the nuclei with S1 buffer (pour 0.5 mL down the side of each well, just for a few seconds).</li>
	<li>Incubate the nuclei with S1 buffer with S1 nuclease (20 U/mL) or without (as a control) for 30 min at 37 &#176;C&#160;(cell incubator).</li>
	<li>Aspirate the S1 buffer and add 0.1% BSA in PBS.</li>
	<li>Scrape the nuclei and transfer them to an appropriately annotated 1.5 mL tube. Keep tubes on ice all the time. 
	NOTE: Successful detachment of nuclei can be checked under a brightfield microscope.</li>
	<li>Pellet the nuclei: centrifuge at ~4600 x g for 5 min, at 4 &#176;C. 
	&#8203;NOTE: Different from the standard DNA fiber assay, the nuclei cannot be counted, so consider the initial number of cells plated. Alternatively, an extra well that was not subjected to permeabilization can be used to estimate the cell count using a hemocytometer or a cell counter.</li>
	<li>Resuspend the pellets well in PBS at a concentration of ~1500 cells/&#181;L, place the tubes on ice and proceed to DNA fiber preparation by spreading (section 4) protocol immediately</li>
</ol>
3. Gap-filling (PRR) experimental setup (3 days)
<ol>
	<li>Day 1: Plate 2 wells for each cell line: &#177; UVC with cells at ~70 % confluency.</li>
	<li>Day 2: Prepare fresh IdU at 20 &#181;M in pre-warmed (37 &#176;C) cell culture media.</li>
	<li>Irradiate the cells with 20 J/m2 UVC.</li>
	<li>Immediately add the media with 20 &#181;M IdU and incubate the cells at 37 &#176;C, 5% CO2 (cell incubator) for precisely 60 min.</li>
	<li>Wash the cells with pre-warmed (37 &#176;C) PBS twice.</li>
	<li>Immediately add the media with 200 ng/mL nocodazole and incubate the cells at 37 &#176;C, 5% CO2 (cell incubator) for 12-24 h (timing of this step must be kept consistent between experiments). 
	&#8203;NOTE: The concentration of nocodazole might need to be adapted according to the cell line used. If using a genotoxic drug, incubate the cells with IdU &#177; drug28.</li>
	<li>Day 3: Aspirate the culture media, add the media with 20 &#181;M CldU + nocodazole, and incubate for the last 4 h of nocodazole treatment, at 37 &#176;C, 5% CO2 (cell incubator).</li>
	<li>Wash the cells with pre-warmed (37 &#176;C) PBS.</li>
	<li>Add trypsin and incubate for 1-2 min at 37 &#176;C, 5% CO2 (cell incubator) to detach cells.</li>
	<li>Add the same volume of cell culture media and collect cells (media + trypsin) in an appropriately annotated 1.5 mL tube. Keep the tubes on ice all the time.</li>
	<li>Count the cells using a hemocytometer or a cell counter.</li>
	<li>Pellet the cells: centrifuge at ~350 x g for 5 min, at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the cells in PBS at ~1500 cells/&#181;L. Keep the cells on ice and start DNA fiber preparation by spreading protocol immediately (Day 3 or 4).</li>
</ol>
4. DNA fiber preparation by spreading (~1 h for 12 slides)
<ol>
	<li>Annotate the microscope slides with a carbon pencil and place them flat on a tray.</li>
	<li>Tap the bottom of the tube to ensure homogenization.</li>
	<li>Add 2 &#181;L of cells on the top of the corresponding slide. 
	NOTE: Two drops can be added, one on the top of the slide and another in the middle of the slide.</li>
	<li>Add 6 &#181;L of the lysis buffer and lyse the cells by pipetting up and down about 5 times (avoid making bubbles).</li>
	<li>Pull the drop a little bit towards the bottom of the slide with the pipette tip (to guide the path of the drop spreading).</li>
	<li>Incubate for 5 min at RT to lyse the nuclei. 
	NOTE: The volume of lysis buffer and the time of lysis can be adjusted if the lysate dries too quickly or, alternatively, does not dry enough. Lysis buffer volume can vary from 5-7 &#181;L and timing can fall between 4-10 min.</li>
	<li>Tilt the slide at around 20-40&#176; and allow the drop to spread to the bottom of the slide at a constant, low speed.</li>
	<li>Allow the slide to dry at RT in the dark (10-15 min).</li>
	<li>Freshly prepare the fixing solution: Mix methanol: glacial acetic acid at a 3:1 ratio.</li>
	<li>Immerse the slides in a jar containing the fixing solution and incubate for 5 min at RT to fix DNA onto the slides. 
	&#8203;CAUTION: Methanol is highly toxic and should be kept under a chemical hood. Discard in an appropriate container.</li>
	<li>Dry the slides at RT in the dark and store at 4 &#176;C protected from light until staining.</li>
</ol>
5. Immunostaining of DNA fibers (~6 h)
<ol>
	<li>Wash the slides with PBS twice for 5 min.</li>
	<li>Denature the DNA with freshly prepared 2.5 M HCl for 40-60 min at RT. 
	CAUTION: To prepare 2.5 M HCl, pour acid in water and never the opposite. This is an exothermic reaction so it will warm up slightly.</li>
	<li>Wash the slides with PBS three times for 5 min.</li>
	<li>Block with 5% BSA (can be stored at 20 &#176;C for up to three uses) previously warmed at 37 &#176;C for 30-60 min at 37 &#176;C.</li>
	<li>Remove the excess liquid from slides by gently tapping on a paper.</li>
	<li>Add 30 &#181;L of primary antibodies (mouse anti-BrdU 1/20 (for IdU) and rat anti-BrdU 1/100 (for CldU) diluted in PBS-T-BSA) to the slides dropwise and place a coverslip on top. Incubate for 1-1.5 h at RT in a dark, humid chamber.</li>
	<li>Place the slides in a jar with PBS for 1-2 min, then carefully remove the coverslips.</li>
	<li>Wash with PBS-T in a jar three times for 5 min, then place the slides in PBS.</li>
	<li>Remove the excess liquid by gently tapping on a paper.</li>
	<li>Add 30 &#181;L of secondary antibodies (anti-mouse Alexa Fluor 594 and anti-rat Alexa Fluor 488 diluted at 1/75 in PBS-T-BSA.) to the slides dropwise and place a coverslip on top. Incubate for 45-60 min at RT in a dark, humid chamber.</li>
	<li>Place the slides in a jar with PBS for 1-2 min, then carefully remove the coverslips.</li>
	<li>Wash with PBS-T in a jar three times for 5 min, then place the slides in PBS.</li>
	<li>Remove the excess liquid by gently tapping on a paper.</li>
	<li>Add 20 &#181;L of the mounting reagent dropwise to the slides and place a coverslip on top. Avoid bubbles.</li>
	<li>Let the slides dry at RT in the dark, then store at 4 &#176;C (or -20 &#176;C for longer conservation).</li>
</ol>
6. Image acquisition and analysis (~1 h per sample)
NOTE: An epifluorescence microscope can be used for both protocols. However, for the gap-filling (PRR) assay, using a confocal microscope is highly recommended for the increased resolution of the images.
<ol>
	<li>Place a drop of immersion oil (microscope specific) near the top of the slide where the cells were lysed and find the focus using the green channel by searching green dots in the background (40x, 63x, and 100x objectives can be used).</li>
	<li>Find the main concentration of fibers and move to the edges to find well-spread non-overlapping individual fibers using only one channel to select the regions for the pictures and avoid potential bias.</li>
	<li>Take around 15-20 pictures per slide alongside the entire slide at the green and red channels and merge the two channels to obtain bicolor DNA fibers. 
	NOTE: If using a confocal microscope, take pictures at high resolution (1024 x 1024 pixels) and use pinhole values close to 1 for both red and green fluorescence.</li>
	<li>Use ImageJ (free software provided by the NIH,&#160;https://imagej.nih.gov/ij/) for IdU and CldU tract length measurements.
	<ol>
		<li>Open a picture by dragging it into the ImageJ box.</li>
		<li>Use the scale bar generated by the microscope software (appropriately calibrated) to convert the length to micrometers. Use the Straight-line tool by selecting the 5th icon from left to right on the ImageJ box to measure the scale bar in the picture provided micrometers and obtain a measurement in pixels. Click on Analyze &#62; Set Scale and replace the &#34;distance in pixels&#34; with the appropriate number and the &#34;unit of length&#34; to &#181;m.</li>
		<li>On the ImageJ box, select the Segmented Line tool by left-clicking on the 5th icon from left to right and then selecting the second option from top to bottom.</li>
		<li>Draw the exact path of the red (IdU) or green (CldU) tract by left-clicking and stop the drawing by right-clicking. Push the M button on the keyboard to measure the length.</li>
		<li>For the S1 Fiber, only analyze red-green (bicolor fibers)16. Measure red and green tract lengths from at least 150 fibers from different pictures by keeping track of the red and green tracts measurements for each individual fiber.</li>
		<li>For the gap-filling assay, only measure the length of IdU (red) tracts that contain at least 1 CldU (green) patch but no continuous CldU staining. Score at least 10-15 IdU tracts from different pictures with at least 1 CldU patch. 
		NOTE: Other software can be used for the analyses, such as Zeiss LSM Image Browser.</li>
	</ol>
	</li>
	<li>For the S1 Fibers, plot the IdU tracts length, CldU tracts length, and ratios (CldU/IdU or vice-versa) in separate graphics as scatter dot plots with medians.</li>
	<li>Measure gap-filling events as units of &#34;per-kilobase density&#34;, divide the total number of CldU (PRR, green) patches on a given length of IdU (red) tract by the total length of the red tract in kilobases (considering 1 &#181;m = 2.59 kilobases31). Then plot the data as scatter dot plots with medians.</li>
	<li>In both experiments, perform statistical analysis between two samples using Mann-Whitney (nonparametric test) and between more than two samples using Kruskal-Wallis followed by Dunn&#39;s multiple comparisons test.</li>
</ol>

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I., Meneghini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+replication+fork+in+ultraviolet+light-irradiated+human+cells.">Structure of the replication fork in ultraviolet light-irradiated human cells.</a> Biophysical Journal. 27 (2), 287-300 (1979).</li><li>Lehmann, A. R., Fuchs, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaps+and+forks+in+DNA+replication:+Rediscovering+old+models.">Gaps and forks in DNA replication: Rediscovering old models.</a> DNA Repair (Amst). 5 (12), 1495-1498 (2006).</li><li>Daigaku, Y., Davies, A. A., Ulrich, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-dependent+DNA+damage+bypass+is+separable+from+genome+replication.">Ubiquitin-dependent DNA damage bypass is separable from genome replication.</a> Nature. 465 (7300), 951-955 (2010).</li><li>Karras, G. I., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RAD6+DNA+damage+tolerance+pathway+operates+uncoupled+from+the+replication+fork+and+is+functional+beyond+S+phase.">The RAD6 DNA damage tolerance pathway operates uncoupled from the replication fork and is functional beyond S phase.</a> Cell. 141 (2), 255-267 (2010).</li><li>Tirman, S., 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=Temporally+distinct+post-replicative+repair+mechanisms+fill+PRIMPOL-dependent+ssDNA+gaps+in+human+cells.">Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells.</a> Molecular Cell. 81 (19), 4026-4040 (2021).</li><li>Cong, 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=Replication+gaps+are+a+key+determinant+of+PARP+inhibitor+synthetic+lethality+with+BRCA+deficiency.">Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.</a> Molecular Cell. 81 (15), 3128-3144 (2021).</li><li>Quinet, 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=PRIMPOL-mediated+adaptive+response+suppresses+replication+fork+reversal+in+BRCA-deficient+cells.">PRIMPOL-mediated adaptive response suppresses replication fork reversal in BRCA-deficient cells.</a> Molecular Cell. 77 (3), 461-474 (2020).</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>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=Visualization+of+DNA+replication+in+the+vertebrate+model+system+DT40+using+the+DNA+fiber+technique.">Visualization of DNA replication in the vertebrate model system DT40 using the DNA fiber technique.</a> Journal of Visualized Experiments: JoVE. (56), e3255 (2011).</li><li>Simoneau, A., Xiong, R., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trans+cell+cycle+effects+of+PARP+inhibitors+underlie+their+selectivity+toward+BRCA1/2-deficient+cells.">The trans cell cycle effects of PARP inhibitors underlie their selectivity toward BRCA1/2-deficient cells.</a> Genes &amp; Development. 35 (17-18), 1271-1289 (2021).</li><li>Taglialatela, 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=REV1-Pol&#950;+maintains+the+viability+of+homologous+recombination-deficient+cancer+cells+through+mutagenic+repair+of+PRIMPOL-dependent+ssDNA+gaps.">REV1-Pol&#950; maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.</a> Molecular Cell. 81 (19), 4008-4025 (2021).</li><li>Lim, K. S., 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=USP1+Is+required+for+replication+fork+protection+in+BRCA1-deficient+tumors.">USP1 Is required for replication fork protection in BRCA1-deficient tumors.</a> Molecular Cell. 72 (6), 925-941 (2018).</li><li>Peng, M., 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=Opposing+roles+of+FANCJ+and+HLTF+protect+forks+and+restrain+replication+during+stress.">Opposing roles of FANCJ and HLTF protect forks and restrain replication during stress.</a> Cell Reports. 24 (12), 3251-3261 (2018).</li><li>Huang, 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+DNA+translocase+FANCM/MHF+promotes+replication+traverse+of+DNA+interstrand+crosslinks.">The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks.</a> Molecular Cell. 52 (3), 434-446 (2013).</li><li>Ding, W., Bishop, M. E., Lyn-Cook, L. E., Davis, K. J., Manjanatha, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+alkaline+comet+assay+and+enzyme-modified+alkaline+comet+assay+for+measuring+DNA+strand+breaks+and+oxidative+DNA+damage+in+rat+liver.">In vivo alkaline comet assay and enzyme-modified alkaline comet assay for measuring DNA strand breaks and oxidative DNA damage in rat liver.</a> Journal of Visualized Experiments: JoVE. (111), e53833 (2016).</li><li>Lu, Y., Liu, Y., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+in+vitro+DNA+damage+using+comet+assay.">Evaluating in vitro DNA damage using comet assay.</a> Journal of Visualized Experiments: JoVE. (128), e56450 (2017).</li></ol>

The authors have nothing to disclose.

Critical steps of the standard DNA fiber assay protocol were discussed in a previous publication32. Here, we describe modified versions of the standard DNA fiber assay to investigate the presence of post-replicative ssDNA gaps as well as their repair by gap-filling, initially described in14. In the context of post-replicative ssDNA gap presence, the use of the S1 nuclease in the S1 Fiber protocol would most likely be suitable after exposure of a genotoxic agent for a minimu...

Seminal works have provided evidence of the accumulation of post-replicative single-stranded (ssDNA) gaps upon treatment with DNA damaging agents in bacteria1 and human cells2,3. During replication of damaged DNA templates, the DNA synthesis machinery may bypass the lesions by employing specific translesion synthesis DNA polymerases or through template switching mechanisms. Alternatively, the replisome may also simply skip the lesion leaving an ssDNA gap behind, to be repaired later. More recently, a study clearly showed that treatment with genotoxic agents leads to ssDNA gaps in eukaryotes by utilizing electron microscopy to visualize the specific structure of replication intermediates4. The formation of these regions of post-replicative gaps was initially proposed to be a simple result of the semi-discontinuous mode of DNA replication2. In this case, a lesion on the lagging strand can block the elongation of an Okazaki fragment, but fork progression is naturally rescued by the following Okazaki fragment, leaving behind an ssDNA gap. However, further studies demonstrated that the formation of gaps on the leading strand is also possible, as first shown in bacteria5. In eukaryotes, PRIMPOL, a unique DNA polymerase with primase activity, was shown to be able to restart DNA synthesis downstream a replication blocking lesion through its repriming activity6,7,8,9,10,11. Thus, the PRIMPOL primase activity may explain the formation of post-replicative ssDNA gaps in the leading strand upon treatment with a DNA damaging agent in human cells12. Nonetheless, detection of these gaps as well as gap repair, until recently, required indirect or time-consuming approaches such as electron microscopy4 or plasmid-based assays13. The use of the ssDNA-specific S1 nuclease to detect gaps in human cells was pioneered by early studies more than forty years ago, using sucrose gradient techniques2,3. More recently, our group applied the use of this nuclease to detect ssDNA in replicating DNA (post-replicative gaps) using other methods such as DNA fiber and comet assays14. These new approaches paved the way for the current surge of studies on post-replicative gaps. Here, we describe a strategy of using S1 nuclease to detect post-replicative ssDNA gaps by DNA fiber assay and explain how a differential labeling scheme in the DNA fiber protocol can permit the study of the repair of these gaps.
The DNA fiber assay is a powerful technique that has been used by a growing number of labs and has provided valuable insights into replication fork dynamics and replication stress response mechanisms. Briefly, this technique is based on the sequential incorporation of nucleotide analogs (such as CldU -5-chloro-2&#39;-deoxyuridine-, and IdU -5-iodo-2&#39;-deoxyuridine) in the replicating DNA. After harvesting, cells are lysed, and DNA molecules spread on a positively coated glass slide. CldU and IdU are then detected by specific antibodies, which can be visualized in a fluorescent microscope as bicolored fibers. Finally, the lengths of the IdU and CldU tracts are measured to identify any alterations of DNA replication dynamics as a consequence of DNA damage induction. This technique can be utilized to investigate different phenomena, such as fork stalling, fork slowing, nascent DNA degradation, and variations in the frequency of origin firing15,16.
One limitation of the DNA fiber assay is its resolution of a few kilobases. Because post-replicative ssDNA gaps can be in the range of hundreds of bases, it is impossible to visualize these gaps directly by the standard DNA fiber protocol. The presence of ssDNA gaps on replicating DNA in human cells treated with genotoxic agents has been indirectly implicated before. For example, the observation of ssDNA as assessed by recruitment of the ssDNA-binding protein, replication protein A (RPA), in cells outside the S phase, or the formation of ssDNA as detected by alkaline DNA unwinding combined with the absence of prolonged fork stalling by DNA fiber assay12,17,18,19,20,21 was attributed to the accumulation of ssDNA gaps. In addition, post-replicative ssDNA gaps induce an ATR-dependent G2/M phase checkpoint and arrest cells treated with replication poisons18,19,20,21,22.
The S1 nuclease degrades single-stranded nucleic acids releasing 5&#39;-phosphoryl mono- or oligonucleotides and has a 5-times higher affinity to ssDNA compared to RNA. Double-stranded nucleic acids (DNA:DNA, DNA:RNA, or RNA:RNA) are resistant to the S1 nuclease except when used in extremely high concentrations. The S1 nuclease also cleaves double-stranded DNA at the single-stranded region caused by a nick, gap, mismatch, or loop. The S1 nuclease is thus an ssDNA-specific endonuclease capable of cleaving ssDNA gaps, ultimately generating double-stranded breaks23,24. Thus, adding steps for S1 nuclease digestion to the DNA fiber protocol indirectly enables the detection of post-replicative ssDNA gaps. In the presence of gaps, treatment of exposed nuclei with the S1 nuclease prior to DNA spreading will generate shorter tracts as a consequence of S1 cleavage of ssDNA inherently present at gaps14. Accordingly, tract shortening is the read-out for ssDNA gaps using this approach. Compared to the standard DNA fiber protocol, the DNA fiber with the S1 nuclease only requires two extra steps: nuclei exposure (cell permeabilization) and treatment with the S1 nuclease. It is important to note that appropriate controls are mandatory, such as samples treated with the genotoxic agent but without the S1 nuclease and samples treated with the S1 nuclease without the genotoxic agent. The protocol itself, including the incorporation of analogs, S1 treatment, and spreading, can be performed in one day and does not require exceptional material. It only requires the thymidine analogs, the purified S1 nuclease, the appropriate primary and secondary antibodies, and a fluorescent microscope. Overall, the DNA fiber employing the S1 nuclease detects ssDNA gaps on ongoing replication forks using a relatively simple approach.
The post-replicative ssDNA gaps formed as a consequence of replication stress response mechanisms can be repaired (or filled) by different mechanisms, including translesion DNA synthesis or template switching, in a process called gap-filling or post-replication repair (PRR)25. These processes occur behind the advancing forks, involving replication-independent DNA synthesis14,26,27. Based on these findings, a labeling scheme distinct from the standard DNA fiber assay can be performed to visualize gap-filling events in the G2 phase directly14,16,26,28. Specifically, one thymidine analog can be used to label the replication fork at the time of genotoxic treatment and post-replicative ssDNA gap formation, while another thymidine analog can be used to label gap-filling events. In this protocol, cells are labeled with a first thymidine analog (IdU, for example) immediately after or concomitantly with genotoxic treatment for 1 h, so that the nascent DNA is labeled at the time of gap formation. Nocodazole is added upon treatment for anywhere between 12-24 h to arrest cells in G2/M, preventing the following S phase. For the last 4 h of nocodazole treatment, a second thymidine analog (CldU, for example) is added to the media to be incorporated during gap filling. Importantly, this assay can only be used to detect gap-filling events in G2 because the gap-filling signal from CldU cannot be distinguished from a signal due to CldU incorporation into replicating DNA in the S phase. Therefore, to minimize the background signal, the timing of CldU incorporation after damage induction should coincide with when most of the cell population is entering the G2 phase14. Therefore, this timing will vary depending on cell line and treatment conditions. Optimizing cell cycle progression prior to employing this assay is advised. Co-staining of these thymidine analogs allows the visualization of gap-filling (PRR) tracts (CldU patches) on top of nascent DNA (IdU tracts) synthesized during the genotoxic treatment when ssDNA gaps were generated.

<table><tbody><tr><td>Acetic acid, Glacial</td><td>Synth</td><td>64-19-7</td><td>Alternatively, BSA - Biosera - REF PM-T1725/100</td></tr><tr><td>Ammonium hydroxide</td><td>Synth</td><td>1336-21-6</td><td>Or similar</td></tr><tr><td>Antibody anti-mouse IgG1 Alexa Fluor 594</td><td>Invitrogen</td><td>A11005</td><td>-</td></tr><tr><td>Antibody anti-rat Alexa Fluor 488</td><td>Invitrogen</td><td>A21470</td><td>-</td></tr><tr><td>Antibody Mouse anti-BrdU</td><td>Becton Disckson</td><td>347580</td><td>-</td></tr><tr><td>Antibody Rat anti-BrdU</td><td>Abcam</td><td>&amp;nbsp;Ab6326</td><td>-</td></tr><tr><td>Biological security hood</td><td>Pachane</td><td>PA 410</td><td>Use hood present in the laboratory</td></tr><tr><td>BSA (Bovine Serum Albumin)</td><td>Sigma-Aldrich</td><td>A3294</td><td>Or similar</td></tr><tr><td>Cell scraper</td><td>Thermo Scientific</td><td>179693</td><td>Or similar</td></tr><tr><td>CldU</td><td>Millipore-Sigma</td><td>C6891</td><td>-</td></tr><tr><td>Cloridric acid</td><td>Synth</td><td>7647-01-0</td><td>Or similar</td></tr><tr><td>Confocal Zeiss LSM Series (7, 8 or 9)</td><td>Zeiss</td><td>-</td><td>Or similar</td></tr><tr><td>Cover glass (or coverslips)</td><td>Thermo Scientific</td><td>152460</td><td>Alternatively, Olen - Kasvi Cover Glass (24 x 60 mm) - K5-2460</td></tr><tr><td>DMEM - High Glucose</td><td>LGC/Gibco</td><td>BR30211-05/12100046</td><td>Use culture media specific for the cell line used.</td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid disodium salt dihydrate)</td><td>Sigma-Aldrich</td><td>E5314</td><td>Or similar</td></tr><tr><td>Epifluorescence Microscope Axiovert 200</td><td>Zeiss</td><td>-</td><td>Or similar</td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Gibco</td><td>12657-029</td><td>Or similar</td></tr><tr><td>Forma Series II Water Jacketed CO2 Incubator</td><td>Thermo Scientific 3110</td><td>13-998-074</td><td>Use cell incubator present in the laboratory</td></tr><tr><td>Glass slide jar</td><td>Sigma-Aldrich</td><td>S5516</td><td>Or similar</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>56-81-5</td><td>Or similar</td></tr><tr><td>Idu</td><td>Millipore-Sigma</td><td>I7125</td><td>-</td></tr><tr><td>Magnesium Chloride</td><td>Synth</td><td>7791-18-6</td><td>Or similar</td></tr><tr><td>Methanol</td><td>Merck</td><td>67-56-1</td><td>Or similar</td></tr><tr><td>Microscope slides</td><td>Denville</td><td>M1021</td><td>Alternatively, Olen - Kasvi Microscope Slides - K5-7105 &lt;strong&gt;OR&lt;/strong&gt; Precision Glass Line - 7105-1</td></tr><tr><td>MOPS (&amp;Aacute;cido 3-morfolinopropano 1-sulf&amp;ocirc;nico)</td><td>Synth</td><td>1132-61-2</td><td>Or similar</td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>31430-18-9</td><td>-</td></tr><tr><td>PBS (Phosphate Buffer Saline)</td><td>Life Thechnologies</td><td>3002</td><td>Or similar</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td>Or similar</td></tr><tr><td>ProLong Gold AntiFade Mountant</td><td>Invitrogen</td><td>P36930</td><td>Any antifade moutant solution for immunofluorescence could be used</td></tr><tr><td>S1 nuclease purified from &lt;em&gt;Aspergillus oryzae&lt;/em&gt;</td><td>Invitrogen</td><td>18001-016</td><td>Pre-dilute the S1 nuclease (1/100 - 1/200) in S1 nuclease dilution buffer provided by the manufacturer, aliquote and store at -20 &amp;deg;C</td></tr><tr><td>SDS (Sodium Dodecyl Sulfate)</td><td>BioRad</td><td>161-0302</td><td>Or similar</td></tr><tr><td>Sodium Acetate Trihydrate</td><td>Sigma-Aldrich</td><td>6131-90-4</td><td>Or similar</td></tr><tr><td>Sodium Chloride</td><td>Synth</td><td>&amp;nbsp;7647-14-5</td><td>Or similar</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>57-50-1</td><td>Or similar</td></tr><tr><td>Tris Base</td><td>West Lab Research</td><td>BP152-1</td><td>Or similar</td></tr><tr><td>Triton X-100</td><td>Synth</td><td>9002-93-1</td><td>Or similar</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200072</td><td>Or similar</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td>Or similar</td></tr><tr><td>UVC Lamp</td><td>Non Specific</td><td>-</td><td>Essential: emission lenght of 254 nm</td></tr><tr><td>VLX-3W UV Radiometer</td><td>Vilber Loumart</td><td>-</td><td>Or similar</td></tr><tr><td>Zinc Acetate</td><td>Sigma-Aldrich</td><td>557-34-6</td><td>Or similar</td></tr></tbody></table>

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.

In the S1 Fiber assay, if treatment with a genotoxic agent leads to post-replicative ssDNA gaps, the overall lengths of DNA fibers from S1-treated nuclei will be shorter upon treatment with DNA damage compared to untreated samples as well as to samples that were treated with the genotoxic agent but were not submitted to S1 cleavage (Figure 1).
Alternatively, if treatment with the S1 nuclease does not significantly affect the length of DNA fibers compared to untrea...

Watch this Scientific Journal Video about Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay at JoVE.com

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

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 ...

detection-post-replicative-gaps-accumulation-repair-human-cells-using

Research

JoVE Journal

Genetics

5.8K Views.  University of São Paulo. 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. 

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

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Cancer Research

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

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 ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Biology

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 ...

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

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Investigating the Extent of DNA Breaks Occurring During DNA Replication 

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

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 ...

Bioengineering

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 ...

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). 
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2&#8242;-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...

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.

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.