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 ...

Research

JoVE Journal

Genetics

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

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Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

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Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

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

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Rare Event Detection Using Error-corrected DNA and RNA Sequencing

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Methodology for Accurate Detection of Mitochondrial DNA Methylation

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

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Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

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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 ...