Name: visualization of replisome encounters with an antigen tagged blocking lesion
Uploaded: 2021-07-27T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion at JoVE.com

This research was supported, in part, by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746&#8211;08). J.H. is supported by the National Natural Science Foundations of China (21708007 and 31871365).

1. Cell preparation
<ol>
	<li>Day 1
	<ol>
		<li>Pre-treat 35 mm glass-bottomed culture dishes with a cell adhesive solution.</li>
		<li>Plate cells in the pre-treated dishes one day before treatment. Cell should be actively dividing and 50&#8211;70% confluent on the day of the experiment. 
		NOTE: HeLa cells were used in this experiment with Dulbecco Modified Eagle Medium DMEM, supplemented with 10% fetal bovine serum, 1x penicillin /streptomycin. There is no restriction for adherent cell lines. However, non-adher...

<ol>
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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=Kinetics+and+fidelity+of+the+repair+of+Cas9-induced+double-strand+DNA+breaks.">Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks.</a> Molecular Cell. 70 (5), 801-813 (2018).</li><li>Vitor, A. C., Huertas, P., Legube, G., de Almeida, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+DNA+double-strand+break+repair:+An+ever-growing+toolbox.">Studying DNA double-strand break repair: An ever-growing toolbox.</a> Frontiers in Molecular Bioscience. 7, 24 (2020).</li><li>Galbiati, A., Beausejour, C., d'Adda di, F. 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C., Smogorzewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fanconi+anaemia+and+the+repair+of+Watson+and+Crick+DNA+crosslinks.">Fanconi anaemia and the repair of Watson and Crick DNA crosslinks.</a> Nature. 493 (7432), 356-363 (2013).</li><li>Kaushal, S., Freudenreich, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fork+stalling+and+DNA+structures+in+causing+chromosome+fragility.">The role of fork stalling and DNA structures in causing chromosome fragility.</a> Genes Chromosomes Cancer. 58 (5), 270-283 (2019).</li><li>Knipscheer, P., Raschle, M., Scharer, O. D., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-coupled+DNA+interstrand+cross-link+repair+in+Xenopus+egg+extracts.">Replication-coupled DNA interstrand cross-link repair in Xenopus egg extracts.</a> Methods in Molecular Biology. 920, 221-243 (2012).</li><li>Klein, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPF-ERCC1+acts+in+Unhooking+DNA+interstrand+crosslinks+in+cooperation+with+FANCD2+and+FANCP/SLX4.">XPF-ERCC1 acts in Unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4.</a> Molecular Cell. 54 (3), 460-471 (2014).</li><li>Long, D. T., Raschle, M., Joukov, V., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+RAD51-dependent+DNA+interstrand+cross-link+repair.">Mechanism of RAD51-dependent DNA interstrand cross-link repair.</a> Science. 333 (6038), 84-87 (2011).</li><li>Benedetto, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+psoralens.+An+historical+perspective.">The psoralens. An historical perspective.</a> Cutis. 20 (4), 469-471 (1977).</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>Thazhathveetil, A. K., Liu, S. T., Indig, F. E., Seidman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoralen+conjugates+for+visualization+of+genomic+interstrand+cross-links+localized+by+laser+photoactivation.">Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation.</a> Bioconjugate Chemistry. 18 (2), 431-437 (2007).</li><li>O'Donnell, M. E., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ring-shaped+hexameric+helicases+that+function+at+DNA+replication+forks.">The ring-shaped hexameric helicases that function at DNA replication forks.</a> Nature Structural &amp; Molecular Biology. 25 (2), 122-130 (2018).</li><li>Koos, B., 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=Analysis+of+protein+interactions+in+situ+by+proximity+ligation+assays.">Analysis of protein interactions in situ by proximity ligation assays.</a> Current Topics in Microbiology and Immunology. 377, 111-126 (2014).</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=Remodeling+of+Interstrand+Crosslink+Proximal+Replisomes+Is+Dependent+on+ATR,+FANCM,+and+FANCD2.">Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2.</a> Cell Reports. 27 (6), 1794-1808 (2019).</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=Single+molecule+analysis+of+laser+localized+psoralen+adducts.">Single molecule analysis of laser localized psoralen adducts.</a> Journal of Visualized Experiments. (122), e55541 (2017).</li><li>Saldivar, J. C., Cortez, D., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+kinase+ATR:+ensuring+faithful+duplication+of+a+challenging+genome.">The essential kinase ATR: ensuring faithful duplication of a challenging genome.</a> Nature Reviews Molecular Cell Biology. 18 (10), 622-636 (2017).</li><li>Cortez, D., Glick, G., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minichromosome+maintenance+proteins+are+direct+targets+of+the+ATM+and+ATR+checkpoint+kinases.">Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases.</a> Proceedings of the National Academy of Sciences. 101 (27), 10078-10083 (2004).</li><li>Ersoy, I., Bunyak, F., Chagin, V., Cardoso, M. 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Although the PLA is a very powerful technique, there are technical concerns that must be solved in order to obtain clear and reproducible results. The antibodies must be of high affinity and specificity. Furthermore, it is important to reduce the non-specific background signals as much as possible. We have found that membranes and cellular debris contribute to the background, and we have removed them as much as possible. The washes with detergent containing buffers prior to fixing, and the wash with methanol after fixing...

An erratum was issued for: <a href="https://www.jove.com/t/6517">Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion</a>.
The Authors section was updated from:
Jing Zhang*1 
Jing Huang*2 
Ryan C. James3 
Julia Gichimu1 
Manikandan Paramasivam4 
Durga Pokharel5 
Himabindu Gali6 
Marina A. Bellani1 
Michael M Seidman1 
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health 
2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University 
3Department of Molecular Biology and Genetics, Cornell University 
4Department of Cellular and Molecular Medicine, University of Copenhagen 
5Horizon Discovery 
6Boston University School of Medicine 
* These authors contributed equally
to:
Jing Zhang*1 
Jing Huang*2 
Ishani Majumdar1 
Ryan C. James3 
Julia Gichimu1 
Manikandan Paramasivam4 
Durga Pokharel5 
Himabindu Gali6 
Marina A. Bellani1 
Michael M Seidman1 
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health 
2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University 
3Department of Molecular Biology and Genetics, Cornell University 
4Department of Cellular and Molecular Medicine, University of Copenhagen 
5Horizon Discovery 
6Boston University School of Medicine 
* These authors contributed equally

An erratum was issued for: <a href="https://www.jove.com/t/6517">Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion</a>. The Authors section was updated.

Erratum: Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

The cellular response to double strand breaks is well documented owing to a succession of increasingly powerful methods for directing breaks to specific genomic sites1,2,3. The certainty of location enables unambiguous characterization of proteins and other factors that accumulate at the site and participate in the DNA Damage Response (DDR) thereby driving the Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) pathways that repair breaks. Of course, many breaks are introduced by agents such as radiation and chemical species that do not attack specific sequences4. However, for these there are procedures available that can convert the ends to structures amenable for tagging and localization5,6. Breaks are also introduced by biological processes, such as immunoglobulin rearrangement, and recent technology permits their localization, as well7. The relationship between responding factors and those sites can then be determined.
Breaks also appear as an indirect consequence of adducts formed by compounds that are not inherent breakers but disrupt DNA transactions such as transcription and replication. They may be formed as a feature of the cellular response to these obstructions, perhaps during repair or because they provoke a structure that is vulnerable to nuclease attack. Typically, the physical relationship between the adduct, the break, and the association with responding factors is inferential. For example, ICLs are formed by chemotherapeutics such as cisplatin and Mitomycin C8 and as a reaction product of abasic sites9. ICLs are well known as potent blocks to replication forks10, thereby stalling forks which can be cleaved by nucleases11. The covalent linkage between strands is often relieved by pathways that have obligate breaks as intermediates12,13, necessitating homologous recombination to rebuild the replication fork14. In most experiments the investigator follows the response of factors of interest to the breaks which are formed downstream of the collision of a replication fork with an ICL. However, because there has been no technology for the localization of a provocative lesion, the proximity of the replisome, and its component parts, to the ICL can only be assumed.
We have developed a strategy to enable the analysis of protein associations with non-sequence specific covalent adducts, illustrated here by ICLs. In our system these are introduced by psoralen, a photoactive natural product used for thousands of years as a therapeutic for skin disorders15. Our approach is based on two important features of psoralens. The first is their high frequency of crosslink formation, which can exceed 90% of adducts, in contrast to the less than 10% formed by popular compounds such as cisplatin or Mitomycin C8,16. The second is the accessibility of the compound to conjugation without loss of crosslinking capacity. We have covalently linked trimethyl psoralen to Digoxigenin (Dig), a long established immunotag. This enables detection of the psoralen adducts in genomic DNA by immunostaining of the Dig tag, and visualization by conventional immunofluorescence17.
This reagent was applied, in our previous work, to the analysis of replication fork encounters with ICLs using a DNA fiber-based assay16. In that work we found that replication could continue past an intact ICL. This was dependent on the ATR kinase, which is activated by replication stress. The replication restart was unexpected given the structure of the CMG replicative helicase. This consists of the MCM hetero-hexamer (M) that forms an offset gapped ring around the template strand for leading strand synthesis which is locked by the proteins of the GINS complex (G, consisting of PSF1, 2, 3, and SLD5) and CDC45 (C)18. The proposal that replication could restart on the side of the ICL distal to the side of the replisome collision argued for a change in the structure of the replisome. To address the question of which components were in the replisome at the time of the encounter with an ICL we developed the approach described here. We exploited the Dig tag as a partner in Proximity Ligation Assays (PLA)19 to interrogate the close association of the ICL with proteins of the replisome20.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 568, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody</td>
			<td>Invitrogen</td>
			<td>A-10011</td>
			<td>1 in 1000</td>
		</tr>
		<tr>
			<td>35 mm plates with glass 1.5 coverslip</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</td>
			<td>Glass Bottom Microwell Dishes 35mm Petri Dish Microwell</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488,Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody</td>
			<td>Invitrogen</td>
			<td>A-10001</td>
			<td>1 in 1000</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>SeraCare</td>
			<td>1900-0012</td>
			<td>Blocking solution, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>CDC45 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab126762</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Cell adhesive</td>
			<td>Life Science</td>
			<td>354240</td>
			<td>for cell-TAK solution</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikkon</td>
			<td>Nikon TE2000 spinning disk microscope</td>
			<td>equiped with Volocity software</td>
		</tr>
		<tr>
			<td>Digoxigenin (Dig) antibody (mouse)</td>
			<td>Abcam</td>
			<td>ab420</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Dig-TMP</td>
			<td>synthesized in the Seidman Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Amplification reagents (5&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82010</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ detection reagents</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92007</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ oligonucleotide PLA probe MINUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92004</td>
			<td>anti-mouse MINUS, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ oligonucleotide PLA probe PLUS</td>
			<td>Sigma-Aldrich</td>
			<td>DUO92002</td>
			<td>anti-rabbit PLUS, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ wash buffer A</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82046</td>
			<td>Duolink Wash Buffers, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Duolink in situ wash buffer B</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82048</td>
			<td>Duolink Wash Buffers, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>epifluorescent microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200M microscope</td>
			<td>Equipped with the Axio Vision software packages (Zeiss, Germany)</td>
		</tr>
		<tr>
			<td>Formaldehyde 16%</td>
			<td>Fisher Scientific</td>
			<td>PI28906</td>
			<td>for fix solution</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo</td>
			<td>31873</td>
			<td>Blocking solution, reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>open source</td>
			<td>Cell profiler</td>
			<td>works for analysis of single plane images</td>
		</tr>
		<tr>
			<td>Image analysis software-license required</td>
			<td>Bitplane</td>
			<td>Imaris</td>
			<td>Cell Biology module needed. Can quantify PLA dots/nuclei in image stacks (3D) and do 3D reconstructions</td>
		</tr>
		<tr>
			<td>Ligase (1 unit/&#956;l)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82029</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Ligation reagent (5&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82009</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>MCM2 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab4461</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>MCM5 antibody (rabbit monoclonal)</td>
			<td>Abcam</td>
			<td>Ab75975</td>
			<td>1 in 1000</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Lab ALLEY</td>
			<td>A2076</td>
			<td>pre-cold at -20&#176;C before use</td>
		</tr>
		<tr>
			<td>phosphoMCM2S108 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab109271</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Polymerase (10 unit/&#956;l)</td>
			<td>Sigma-Aldrich</td>
			<td>DUO82030</td>
			<td>reagents need to be stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Prolong gold mounting media with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>PSF1 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>ab181112</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>RNAse A 100 mg/ml</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>reagents need to be stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Statistical analysis and data visualization software</td>
			<td>open source</td>
			<td>R studio</td>
			<td>ggplot2 package for generation of dot plot and box plots</td>
		</tr>
		<tr>
			<td>Statistical analysis and data visualization software-license required</td>
			<td>Systat Software</td>
			<td>Sigmaplot V13</td>
			<td></td>
		</tr>
		<tr>
			<td>TMP (trioxalen)</td>
			<td>Sigma-Aldrich</td>
			<td>T6137_1G</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787_250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416_100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UV box</td>
			<td>Southern New England Ultraviolet</td>
			<td></td>
			<td>Discontinued. See Opsytec UV test chamber as a possible replacement</td>
		</tr>
		<tr>
			<td>UV test Chamber</td>
			<td>Opsytec</td>
			<td>UV TEST CHAMBER BS-04</td>
			<td></td>
		</tr>
		<tr>
			<td>VE-821</td>
			<td>Selleckchem</td>
			<td>S8007</td>
			<td>final concentrtion is 1&#181;M</td>
		</tr>
	</tbody>
</table>

visualization of replisome encounters with an antigen tagged blocking lesion

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins.
Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

PLA of Dig-TMP with replisome proteins 
The structure of the Dig-TMP is shown in Figure 1. The details of the synthesis, in which trimethyl psoralen was conjugated through a glycol linker to digoxigenin, have been discussed previously17,21. Incubation of cells with the compound followed by exposure to 365 nm light (UVA) photoactivates the compound and drives the crosslinking reaction. Slightly more than 90% of a...

Watch this Scientific Journal Video about Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion at JoVE.com

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In ...

This technique facilitates the visualization of replisome antigen-tagged DNA adduct encounters and can be extended to the interactions of any protein with a DNA structure or lesion amenable to immunodetection. This method can be used to assess interaction frequencies within individual cells, rather than across a population, and allows the visualization of cell cycle-dependent interactions in situ without damaging cells. Demonstrating the procedure will be Ishani Majumdar, a post-doc from the Seidman Laboratory.On the day before the experiment, seed 1.5 times 10 to the fifth cells in two milliliters of medium onto 35-millimeter glass bottom culture dishes pre-treated with cell adhesive solution. The next day, replace the supernatant in each dish with 37 degrees Celsius Five micromolar Dig TMP-supplemented medium to the 50 to 70%confluent cell cultures and return the plates to the cell culture incubator for 30 minutes to allow the Dig TMP to equilibrate. While the cells are incubating, prewarm a UV box to 37 degrees Celsius.At the end of the equilibration, transfer the plates to the pre-warmed box to expose the cells to a five-minute three-joule-per-square-centimeter UVA light dose and replace the supernatants with two milliliter of fresh pre-warmed culture medium before returning the plates to the cell culture incubator for one hour. At the end of the incubation, gently wash the cultures with PBS and fix the cells with one milliliter of 0.1%formaldehyde in PBS for five minutes at room temperature. At the end of the incubation, wash the cells one time with PBS before treating the cells two times with 80 microliters of cytoskeleton extraction buffer supplemented with RNase for five minutes at room temperature per treatment to remove the cytoplasm.After the second incubation, wash the cells with two milliliter of PBS three times before fixing the cells with one milliliter of 4%formaldehyde in PBS for 10 minutes at room temperature. After the second fixation, wash the cells three times with PBS as demonstrated, followed by treatment with one milliliter of cold 100%methanol for 20 minutes at minus 20 degrees Celsius. At the end of the incubation, wash the cells three times in two milliliters of PBS, followed by a 10 minute incubation in 80 microliters of five millimolar Triton X-100 at four degrees Celsius.At the end of the incubation, treat the cells with 100 microliters of five millimolar EDTA in PBS, supplemented with one microliter of 100-milligram-per-milliliter of RNase A for 30 minutes at 37 degrees Celsius. At the end of the incubation, wash the cells three times in PBS before storing them in 5%bovine serum albumin and 10%donkey serum in PBS in a humid chamber overnight at four degrees Celsius. To perform a proximity ligation assay, add 40 microliters of the primary antibody solution of interest to the center of each plate and incubate the plates in the humid chamber for one hour at 37 degrees Celsius.At the end of the incubation, wash the cells three times with two milliliters of PBST before adding 40 microliters of freshly prepared PLA probe solution to each dish for a one hour incubation in the cell culture incubator. At the end of the incubation, wash the cells with three 10-minute washes in two milliliters of buffer A on a tilting platform at room temperature. After the last wash, add 40 microliters of freshly prepared ligation mix to each plate for a 30-minute incubation at 37 degrees Celsius, followed by three two-minute washes in buffer A on the tilting platform.After the last wash, add 40 microliters of freshly prepared amplification solution to each plate for a 100-minute incubation at 37 degrees Celsius in a humid chamber. At the end of the incubation, wash the cells six times with fresh buffer B for 10 minutes per wash on the tilting platform. After the last buffer B wash, wash the cells one time with 0.01x buffer B for one minute at room temperature before labeling with the appropriate secondary antibodies in the humid chamber for 30 minutes at 37 degrees Celsius.At the end of the incubation, wash the samples with three 10-minute washes on a tilting platform in PBST at room temperature before mounting them in mounting medium supplemented with DAPI. within four days of mounting, image at least 100 observations per sample or condition cells on an epifluorescence or confocal microscope, using the same exposure settings for both experimental and control samples. The Dig tag can be visualized by immunofluorescence to reveal the presence of interstrand crosslinks throughout the nucleus.To visualize the interaction of replisomes with interstrand crosslinks, PLA can be applied to visualize the frequency of association of MCM5 and the Dig tag, one hour after interstrand crosslink introduction. As replication stress activates ATR kinase, the PLA between pMCM two serine 108 and the Dig tag is also positive. The PLA results from individual nuclei can be presented in a three-dimensional reconstruction allowing the visualization of replisome interstrand crosslink encounters throughout the nucleus.Replication Fork analysis indicates that interstrand crosslink encounters demonstrate an unexpected replication restart phenomenon, as the proteins of the GINS complex fail to exhibit PLA signal with the interstrand crosslinks. In contrast, the assay with CD 45 is positive, indicating that the other locking protein is retained. When cells are incubated with an ATR inhibitor, the restart is completely suppressed and the GINS Dig PLAs strongly positive.CSK+R treatment is essential for reducing background signal. Be sure to coat the plates with a cell adhesive and to prefix with 0.1%FA or the cells will peel off the plate.

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