Name: detection of post-replicative gaps accumulation and repair in human cells using the dna fiber assay
Uploaded: 2022-02-03T14:15:00
Description: 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

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 ...

<ol>
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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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 OR Precision Glass Line - 7105-1</td>
		</tr>
		<tr>
			<td>MOPS (&#193;cido 3-morfolinopropano 1-sulf&#244;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 Aspergillus oryzae</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 &#176;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>&#160;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 ...

Our protocol opens new possibilities to investigate how replication occurs on damaged DNA. The techniques can detect the formation and repair of single-stranded DNA gaps, opposite the DNA lesion. The S1 nuclease cleaves the single-stranded DNA and allows the detection of post-replicative gaps.This approach was first used by Dr.Meneghini and Dr.Cordiero-Stone in the 1970s in Brazil, and recently adapted by us to the DNA fiber assay. Using the technique described here to study the formation and repair of post-replicative gaps can provide insights into the mechanism of DNA repair and replication stress response that maintain genome integrity. For both protocols described here, the S1 fiber and the gap-filling assays, It is crucial to include all the controls to ensure accurate interpretation of the data.The procedure will be demonstrated by Davi J.Martins, a PhD student from our lab, and Dr.Annabel Quinet, a collaborator who previously developed these techniques when working in our group. To begin with, aspirate the culture media and immediately add the culture media with 20-micromolar of IDU media to the cells. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for precisely 20 minutes.Then, quickly wash the cells with pre-warmed PBS twice and irradiate them with 20 joules per meter square UV-C. Use untreated cells as controls. Immediately add the media with 200-micromolar CldU to the cells and incubate them at 37 degrees Celsius and 5%carbon dioxide for precisely 60 minutes.After that, wash the cells with pre-warmed PBS twice and permeabilize them with the CSK 100 buffer for two to 10 minutes according to the cell line at room temperature. After CSK permeabilization, the nuclei with almost no cytoplasm can be seen compared to those before the permeabilization. Carefully pour PBS down the side of each well to wash the nuclei with PBS.Then, wash the nuclei with S1 buffer. Incubate the nuclei in the S1 buffer with 20 units per milliliter S1 nuclease, and without the S1 nuclease, for 30 minutes at 37 degrees Celsius. Next, aspirate the S1 buffer and add 0.1%BSA in PBS.Scrape the nuclei and transfer them to an approximately annotated 1.5-milliliter tube. Keep the tubes on ice all the time. Centrifuge the nuclei at around 4, 600 times G for five minutes at four degrees Celsius.Resuspend the pellets in PBS at a concentration of approximately 1, 500 cells per microliter. Place the tubes on ice and immediately proceed to DNA fiber preparation by spreading protocol. Annotate the microscope slides with a carbon pencil and place them flat on a tray.Tap the bottom of the tube to ensure homogenization and add two microliters of cells on the top of the corresponding slide. Add six microliters of the lysis buffer and lyse the cells by pipetting up and down about five times. Use the pipette tip to pull the drop slightly towards the bottom of the slide and incubate for five minutes at room temperature to lyse the nuclei.Tilt the slide at around 20 to 40 degrees and allow the drop to spread to the bottom of the slide at a constant low speed. Allow the slide to dry at room temperature in the dark for 10 to 15 minutes. Then, immerse them in a jar containing the freshly prepared fixing solution and incubate for five minutes at room temperature to fix DNA onto the slides.Wash the slides with PBS twice for five minutes and denature the DNA with freshly prepared 2.5-molars cloridric acid for 40 to 60 minutes at room temperature. Wash the slides with PBS three times for five minutes and block with 5%BSA previously warmed for 30 to 60 minutes at 37 degrees Celsius. Gently tap on the paper to remove the excess liquid from the slides.Add 30 microliters of primary antibodies to the slides dropwise and place a cover slip on top. Incubate for 1 to 1.5 hours at room temperature in a dark, humid chamber. Place the slides in a jar with PBS for one to two minutes and then carefully remove the cover slips.Wash with PBST in a jar three times for five minutes and place the slides in PBS. Remove the excess liquid by gently tapping on a paper and add 30 microliters of the secondary antibodies to the slides dropwise. Place a cover slip on top.Incubate for 45 to 60 minutes at room temperature in a dark and humid chamber. Repeat the steps for washing the slides and removing the excess PBS once more and add 20 microliters of the mounting reagent dropwise to the slides. Place a cover slip on top and avoid bubbles.Place a drop of immersion oil near the top of the slide where the cells were lysed. Use the green channel and search the green dots in the background to find the focus. Open the Leica Application Suite software, click on Acquire, and select the green channel.Adjust the settings of the green channel before analysis. Then, change to the red channel and adjust the settings. Find the main concentration of fibers and move to the edges to find the well spread, non-overlapping, individual fibers.Use only one channel to select the regions for the pictures and avoid potential bias. Click on the Acquire Overlay icon and adjust the software for the correct microscope magnification. Take around 15 to 20 pictures per slide alongside the entire slide at the green channel and then at the red channel.The two channels are now merged and the created overlay is moved to a unique folder. It is possible to see the fibers with red and green colors. Open the ImageJ software, left click on the fifth icon, select the second option, and then select the Segmented Line tool.Left click to draw the exact path of the red or green tract and right click to stop the drawing. Push the M button on the keyboard to measure the length. For the S1 fiber, keep track of the red and green tracts measurements for each fiber and measure red and green tract lengths from at least 150 fibers from different pictures.An S1 fiber is shown in these representative images. Control-nascent DNA tract without the gaps and impervious to cleavage by the S1 nuclease is shown here. DNA tract positive for gaps cleaved by the S1 nuclease resulted in a shorter CldU tract length.A differential labeling scheme extensively described in the text manuscript can be performed to label gap-filling events. In this assay, named gap-filling assay, longer IDU tracks with at least one CldU patch or fewer CldU patches correspond with low gap-filling density. Shorter IDU tracts, with at least one CldU patch, and longer IDU tracts with more CldU patches, correspond with high gap-filling density.The permeabilization step of this protocol is crucial for the S1 nuclease to act on the gaps formed during the replication of damaged DNA. The S1 nuclease can also be used in the comet assay protocol to detect the gaps formed during the replication of damaged DNA that persists after treatment. The S1 fiber assay paved the way for the new paradigm that post-replicative gaps can cause cancer vulnerability, particularly in breast and ovarian cancers carrying mutations in BRCA1 and 2 genes.

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Research

JoVE Journal

Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...