Name: visualization of replisome encounters with an antigen tagged blocking lesion
Uploaded: 2021-07-27T13:00:00.000+00:00
Duration: 8 min 24 s
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-adherent cells must be centrifuged onto slides and fixed prior to the analysis by PLA.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Prepare a stock solution of Digoxigenin Trimethyl psoralen (Dig-TMP) by resuspending a frozen aliquot of previously synthesized Dig-TMP in 1:1 EtOH:H2O. Determine the concentration by measuring OD at 250 nm of a 100x dilution (in H2O) of the dissolved Dig-TMP. The extinction coefficient of Dig-TMP is 25,000. Verify the concentration by measuring OD at 250 nm before each use and calculate stock concentration: Abs x 100 x 106/25,000 = Concentration (in &#181;M). Generally, the stock solution is around 3 mM. The solution can be stored in &#8211;20 &#176;C for about a month. 
		NOTE: Dig-TMP must be chemically synthesized in advance, following the procedure described here. Reflux 4&#39;-chloromethyl-4,5&#39;,8-trimethylpsoralen with 4,7,10-trioxa-1,13-tridecanedi-amine in toluene under nitrogen for 12 h. Remove the solvent and recover the 4&#39;-[N-(13-amino-4,7,10-trioxatrideca)] aminomethyl-4,5&#39;,8-trimethylpsoralen product by silica gel chromatography. Conjugate the product to digoxigenin NHS ester in dimethyl formamide and triethylamine at 50 &#176;C for 18 h. Remove the solvent and purify the residue by preparative thin layer silica gel chromatography. Elute the product band chloroform: methanol: 28% ammonium hydroxide (8:1:0.1) mixture. Evaporate the solvents and dissolve the pellet in 50% EtOH:H2O.</li>
		<li>Add Dig-TMP stock in 50% EtOH:H2O to the cell culture medium to a final concentration of 5 &#181;M. Bring the medium to 37 &#176;C. Aspirate the medium from the plates, add the pre-warmed Dig-TMP containing media, and place plates in an incubator (37 &#176;C, 5% CO2) for 30 min to allow the Dig-TMP to equilibrate.</li>
		<li>While the cells are incubating, pre-warm the UV box (see Table of Materials) to 37 &#176;C.</li>
		<li>Place the plates in the pre-warmed UV box and expose the cells to a dose of 3 J/cm2 of UVA light for 5 min for this experiment. Plates were placed on top of a thermo-block maintained at 37 &#176;C, during irradiation. Calculate the time using the formula: 
		<img alt="Equation 1" src="/files/ftp_upload/61689/61689equ01.jpg" /></li>
		<li>Aspirate the medium using a pipette, add fresh pre-warmed medium and place plates back in the incubator at 37 &#176;C, 5% CO2 for 1 h.</li>
		<li>Remove media and wash dishes once gently with phosphate buffered saline (PBS).</li>
		<li>Remove PBS and add 0.1% formaldehyde (FA) in PBS for 5 min at room temperature (RT). This prevents cell detachment during CSK-R (cytoskeleton extraction buffer containing RNase, described in 1.2.9.) pretreatment required to extract the cytoplasmic elements and reduce PLA background.</li>
		<li>Aspirate off FA and wash dishes with PBS once.</li>
		<li>Add CSK-R buffer and incubate for 5 min at RT to remove cytoplasm [CSK-R buffer: 10 mM PIPES, pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5%Triton X-100, 300 &#181;g/mL RNase A]. Aspirate the buffer, add fresh CSK-R, and incubate for 5 min at RT. 
		NOTE: A stock of CSK buffer can be stored at 4 &#176;C, and Triton X and RNaseA added right before use.</li>
		<li>Wash with PBS thrice.</li>
		<li>Fix cells with 4% formaldehyde in PBS for 10 min at RT.</li>
		<li>Wash cells with PBS. Perform this step three times.</li>
		<li>Add cold 100% methanol and incubate for 20 min at -20 &#176;C.</li>
		<li>Wash cells with PBS thrice. At this point cells can be stored in PBS in a humid chamber at 4 &#176;C for up to a week.</li>
		<li>Incubate cells in 80 &#181;L of 5 mM TritonX-100 for 10 min at 4 &#176;C.</li>
		<li>Incubate cells with 100 &#181;L of 5 mM EDTA in PBS supplemented with 1 &#181;L of 100 mg/mL RNase A for 30 min at 37 &#176;C.</li>
		<li>Wash cells with PBS thrice.</li>
		<li>Store cells in blocking buffer (5% BSA and 10% goat serum in PBS) in a humid chamber overnight at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Proximity ligation assay
NOTE: Perform proximity ligation assay on Day 3.
<ol>
	<li>Antibody staining
	<ol>
		<li>Prepare 40 &#956;L of the primary antibody solution per plate: Add the appropriate volume of primary antibodies to achieve desired dilution (mouse anti Digoxigenin and rabbit antibody against a replisome component such as MCM5, CDC45, PSF1 or pMCM2, dilutions specified in Table of Materials) into blocking buffer (to reach a final volume of 24 &#956;L). Mix by tapping and let it stand for 20 min at RT. Prepare a master mix for multiple samples and mix by tapping before applying to the wells.</li>
		<li>Add 40 &#956;L of primary antibody solution to the center of the well and incubate in a humid chamber for 1 h at 37 &#176;C. During staining, allow the PLA probes and blocking buffer to warm to the room temperature.</li>
		<li>Wash cells with PBS-T [PBS-T: 0.05% Tween-20 in 1X PBS] at RT. Perform this step three times.</li>
		<li>While washing, prepare 40 &#956;L of PLA probe solution per dish (PLA probes consist of a secondary antibody recognizing either rabbit or mouse IgG, covalently linked to a PLUS or MINUS oligonucleotide): 8 &#956;L of PLA probe anti mouse-PLUS + 8 &#956;L of PLA probe anti rabbit-MINUS antibody + 24 &#956;L of blocking buffer. Mix and let it stand for 20 min at RT. Prepare a master mix for multiple samples and mix well before applying to the wells. Place the solution in the middle of the well in the plate.</li>
		<li>Remove last wash, add 40 &#956;L of PLA probe solution to the center of the well, and incubate in a humid chamber for 1 h at 37 &#176;C.</li>
		<li>Wash in buffer A thrice, for 10 min each, on a tilting platform at RT. During washing, bring the ligation mix to RT.</li>
	</ol>
	</li>
	<li>Ligation and amplification
	<ol>
		<li>Prepare 40 &#956;L of Ligation mix per plate: 8 &#956;L (5x) of ligation stock + 31 &#956;L of distilled water + 1 &#956;L of ligase. Prepare master mix for multiple plates and mix well before applying to the wells.</li>
		<li>Add 40 &#956;L of ligation solution to each plate and incubate in a humid chamber for 30 min. at 37 &#176;C.</li>
		<li>Wash cells 3x with buffer A, each for 2 min, on a tilting platform at RT.</li>
		<li>Prepare 40 &#956;L of amplification solution per dish: 8 &#956;L (5x) of amplification stock + 31.5 &#956;L of distilled water + 0.5 &#956;L of DNA Polymerase. Prepare a master mix for multiple samples and mix well before applying to the wells.</li>
		<li>Add 40 &#956;L of amplification solution to each plate and incubate in a humid chamber at 37 &#176;C for 100 min.</li>
		<li>Aspirate off the amplification solution and wash with buffer B. Perform 6 washes each for 10 min, on a tilting platform at RT.</li>
		<li>Wash once with 0.01x buffer B for 1 min at RT.</li>
		<li>Aspirate buffer B and incubate plates with secondary antibodies, Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 568 anti-rabbit IgG in blocking solution at appropriate dilutions in blocking buffer, in a humid chamber, for 30 min at 37 &#176;C or overnight at 4 &#176;C.</li>
		<li>Wash cells three times with PBS-T, for 10 min each on a tilting platform at RT.</li>
		<li>Aspirate PBS-T and mount in mounting medium with DAPI. The mounted plates can be imaged immediately or stored at 4 &#176;C in the dark for no more than 4 days before imaging.</li>
	</ol>
	</li>
</ol>
3. Imaging and quantification
<ol>
	<li>Perform imaging in an epifluorescent or confocal microscope (if 3D imaging is desirable, cover at least 3 &#181;m in 15 stacks). Perform experiments in triplicate&#160;and image enough number of fields to make at least 100 observations per sample or condition. Image all fields and samples, including controls, using the same exposure settings.</li>
	<li>Quantify with an appropriate image analysis software (see Table of Materials for open source and commercial software capable of performing this analysis in single or multiple plane-images).
	<ol>
		<li>Segment cell nuclei based on the DAPI staining. Perform detection of nuclear PLA dots. Assign PLA dots to their corresponding nucleus. Export PLA dots per nucleus results as a csv file (see Supplementary File 1 and Supplementary File 2).</li>
	</ol>
	</li>
	<li>Statistical analysis (see Table of Materials for suggested open source and commercial software).
	<ol>
		<li>Verify if the samples follow a normal distribution with a Shapiro-Wilk test.</li>
		<li>Determine whether there is a significant difference between two samples using a Student-t test (if normal distribution assumption met) or a Wilcoxon-Rank Sum test (if normal distribution assumption is violated for a sample).</li>
	</ol>
	</li>
	<li>Data visualization: Generate dot plots combined with box plots to visualize data distribution, median (Q2), 25th (Q1) and 75th percentile for the different samples (See Table of Materials for suggested open source and commercial software).</li>
</ol>
4. 3D display of pMCM2: ICL interactions
<ol>
	<li>Image PLA plates on a spinning disk confocal microscope, using a Plan Fluor 60x/1.25 numerical aperture oil objective. Acquire 16 stacks covering 1.6 mm and generate the 3D reconstruction with the appropriate image analysis software (see Table of Materials).</li>
</ol>

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The authors have nothing to disclose.

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 &amp;deg;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&amp;times;)</td><td>Sigma-Aldrich</td><td>DUO82010</td><td>reagents need to be stored at -20 &amp;deg;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 &amp;deg;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 &amp;deg;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 &amp;deg;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 &amp;deg;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 &amp;deg;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 &amp;deg;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/&amp;mu;l)</td><td>Sigma-Aldrich</td><td>DUO82029</td><td>reagents need to be stored at -20 &amp;deg;C</td></tr><tr><td>Ligation reagent (5&amp;times;)</td><td>Sigma-Aldrich</td><td>DUO82009</td><td>reagents need to be stored at -20 &amp;deg;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&amp;deg;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/&amp;mu;l)</td><td>Sigma-Aldrich</td><td>DUO82030</td><td>reagents need to be stored at -20 &amp;deg;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 &amp;deg;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&amp;micro;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...

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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 ...

visualization-replisome-encounters-with-an-antigen-tagged-blocking

Research

JoVE Journal

Biology

1.3K Views.  National Institutes of Health. Deze techniek vergemakkelijkt de visualisatie van replisoomantigeen-gelabelde DNA-adduct-ontmoetingen en kan worden uitgebreid tot de interacties van elk eiwit met een DNA-structuur of laesie die vatbaar is voor immunodetectie. Deze methode kan worden gebruikt om interactiefrequenties binnen individuele cellen te beoordelen, in plaats van in een populatie, en maakt de visualisatie van celcyclusafhankelijke interacties in situ mogelijk zonder cellen te beschadigen. De procedure wordt gedemonst...

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

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 ...

Genetics

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Cancer Research

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Detection of G1 Phase Single-Stranded DNA Foci via RPA2 Immunofluorescent Staining

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair necessitates collaboration between numerous proteins, responsible for covering a broad range of tasks from recognizing and signaling the presence of a DNA lesion to physically repairing it. During this process, tracks of single-stranded DNA (ssDNA) are often created, which are eventually filled by DNA polymerases. The nature of these ssDNA tracks (in terms of both length and number), along with the polymerase recruited to fill these gaps, are repair pathway-specific. The visualization of these ssDNA tracks can help us understand the complicated dynamics of DNA repair mechanisms.
This protocol provides a detailed method for the preparation of G1 synchronized cells to measure ssDNA foci formation upon genotoxic stress. Using an easy-to-utilize immunofluorescence approach, we visualize ssDNA by staining for RPA2, a component of the heterotrimeric replication protein A complex (RPA). RPA2 binds to and stabilizes ssDNA intermediates that arise upon genotoxic stress or replication to control DNA repair and DNA damage checkpoint activation. 5-Ethynyl-2'-deoxyuridine (EdU) staining is used to visualize DNA replication to exclude any S phase cells. This protocol provides an alternative approach to the conventional, non-denaturing 5-bromo-2'-deoxyuridine (BrdU)-based assays and is better suited for the detection of ssDNA foci outside the S phase.

Author Spotlight: Visualizing Single-Stranded DNA During DNA Repair for Therapeutic Insights 

The following protocol presents the detection of single-stranded DNA foci in the G1 phase of the cell cycle utilizing cell cycle synchronization followed by RPA2 immunofluorescent staining.

DNA has dedicated cellular repair pathways capable of coping with lesions that could arise from both endogenous and/or exogenous sources. DNA repair ...

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion

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