We thank Funda&#231;&#227;o de Amparo a Pesquisa do Estado de S&#227;o Paulo (FAPESP, through Grant Tem&#225;tico 2022/15126-9 to JK and fellowship 21/09439-1 to LARM) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnol&#243;gico (CNPq) for funding this research.

1. Cell plating
NOTE: Cells can be seeded in microscopic fluorescence slides, chambers, or plates. The use of small coverslips or 96/ 384 well plates is recommended for testing multiple conditions and spare reagents. The use of coverslips is advisable for cell lines that detach easily, like HEK293 cells. The coverslips can be previously coated with a poly-L-lysine solution to improve the attachment.
<ol>
	<li>Plate cells considering a vehicle, technical controls, and biological controls. Use technical replicates at two or three wells per condition. 
	NOTE: The use of technical control (both secondaries and one primary only) is mandatory. If possible, biological control should also be included (e.g., overexpression or knockdown of one of the target proteins or a condition that is known to change the interaction, for example).
	<ol>
		<li>Culture U2OS cells in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% of Fetal Bovine Serum (FBS), 4.5 g/L Glucose, and 4 mM of L-Glutamine on tissue culture-treated plates at 37 &#176;C in 5% CO2 and a humidified atmosphere containing 90% air. Depending on the number of conditions to be tested and if using a 384 or coverslips at a 60 mm plate, start from a 60- or 100-mm plate, respectively.</li>
		<li>When the cells reach 60%-70% confluency, split cells using 0.25% trypsin-EDTA 14. Count cells using a Neubauer chamber or an automatic counter14. Plate 4 x 105 cells in 60 mm plate containing multiple 13 mm round coverslips or 4 x 105 cells per well in 384-well plate(s) and incubate for 12 - 24 hours at 37 &#176;C in a humidified, 5% CO2 incubator.</li>
	</ol>
	</li>
</ol>
2. Preparation of solutions
NOTE: The concentration and times of the treatment were chosen based on literature results, which point out the activation of DNA damage repair pathways15,16.
<ol>
	<li>Prepare the stock solution of Etoposide at 50 mM concentration in DMSO. The stock solution can be kept at -20 &#176;C for several months. Dilute the etoposide solution for use in the complete culture medium at 25 &#181;M final concentration. Use DMSO at 0.05% diluted in the complete culture medium as vehicle control.</li>
	<li>Prepare 20x Saline Sodium Citrate (SSC; 3.0 M NaCl, 0.30 M trisodium citrate pH 7.0). This solution can be kept for several months at 4 &#176;C.</li>
</ol>
3. Cell treatment and fixation
NOTE: Do not allow cells to dry at any moment; try to tap off the previous solutions as much as possible. The appropriate method for cell fixation (ice-cold methanol or 4% paraformaldehyde (PFA)) must be determined previously, as well as the antibody dilution, using a conventional immunofluorescence experiment.
CAUTION: Methanol must be disposed of properly.
<ol>
	<li>Remove the culture medium from the wells and add a culture medium containing etoposide or DMSO at 25 &#181;M or 0.05%, respectively. Incubate cells at 37 &#176;C in 5% CO2 and a humidified atmosphere containing 90% air with etoposide or DMSO for 3 different periods of time: 20 min, 1 h, and 3 h. When using 384 plates, all conditions must be fixed simultaneously.
	<ol>
		<li>For this, start by treating the condition with the longest time of treatment, such as 3 h. After 2 h, treat the 1 h treatment condition, and when there is 20 min left for fixation, treat the 20 min condition. Following this, all conditions can be fixed at the same time.</li>
	</ol>
	</li>
	<li>Remove the medium from the wells and wash the cells with PBS 3x.</li>
	<li>Fix the cells by adding ice-cold methanol with enough volume to cover the well surface. Incubate for 10 min at -20 &#176;C. Wash the wells with PBS 3x. 
	NOTE: At this point, the protocol can be paused. The plates can be stored at 4 &#176;C for 1 week.</li>
</ol>
4. Permeabilization and blocking
NOTE: If using the PFA fixation method, a step of blocking with glycine must be performed before permeabilization. The humidity chamber can be prepared by putting a water pan inside the incubator, resulting in a humidity of around 95%. The 384 plate or slides can be incubated in a closed plastic container above filter paper or moistened sponge and transparent film.
<ol>
	<li>Tap off the PBS from the samples.</li>
	<li>Add 30-40 &#181;L of permeabilization buffer (0.2% Triton X-100 in PBS) and incubate for 20 min at room temperature. Wash 3x with PBS</li>
	<li>Tap off the PBS from the samples and add 30-40 &#181;L of 1x blocking solution provided in the PLA kit (alternatively, 3% of Bovine Serum Albumin and Triton X-100 0.1% in PBS solution can be used) to each well/coverslip.</li>
	<li>Incubate the plate in a pre-heated humidity chamber at 37 &#176;C for 1 h.</li>
</ol>
5. Primary antibody incubation (PLA)
NOTE: If using coverslips, it is important to ensure that the antibody solution covers all coverslip surfaces. Special attention must be paid to antibody selection. The antibodies must be from different animal species, and the secondary antibody used for &#947;-H2AX must not be against the species host of the PLA probes production. For example, the primary antibodies used for PLA were raised in mice and goat animals. The PLA probes, anti-mouse, and anti-goat were produced in the donkey. The antibody against &#947;-H2AX was produced in rabbits, and the secondary utilized is anti-rabbit produced in donkeys. In this way, the secondary will not recognize the PLA probes.
<ol>
	<li>Dilute the primary antibodies (the mouse anti-Ku70 1:100 and goat anti-Nek4 1:50; rabbit anti-Nek5 1:25 and mouse anti-TOPII&#946; 1:25) in antibody diluent provided in the kit (alternatively, 3% of Bovine Serum Albumin and Triton X-100 0.1% in PBS solution can be used). Prepare 30 &#181;L of antibody solution per condition if using coverslips or 20 &#181;L per well if using a 384-well plate.</li>
	<li>Tap off the blocking solution. Add the primary antibody solution to each well. Incubate at 4 &#176;C overnight in a humidity chamber.</li>
</ol>
6. PLA probe incubation
NOTE: Choose the probes, taking into consideration not only the primary antibodies but also those intended for use in the immunofluorescence steps. When using 384-well plates for long 37 &#176;C incubation, slow rotation can be used to improve the distribution of the antibody solution.
<ol>
	<li>Dilute the minus and plus probes 1:5 in the antibody diluent. Prepare 10 &#181;L per well if using 384-well plates or 20 &#181;L per sample if using coverslips. Utilize the following combinations: Donkey anti-mouse minus with Donkey anti-goat plus; Donkey anti-mouse minus with Donkey anti-rabbit plus.</li>
	<li>Tap off the primary antibody solution. Wash 1x with TBS-T (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% of Tween-20). Wash 3x for 5 min each with TBS-T at room temperature. Perform an additional&#160;wash with TBS-T.</li>
	<li>Tap off and add the PLA probe solution with 10 &#181;L for the plates and 20 &#181;L for the coverslips. Incubate in the pre-heated humidity chamber at 37 &#176;C for 1 h.</li>
</ol>
7. Ligation
NOTE: Wait to add Ligase until immediately before adding it to the samples. Ligase must be kept in a freezer block (-20 &#176;C). Remove completely all the wash solution before adding the ligase solution. Avoid leaving the cells without a solution for long periods of time. When using 384-well plates for the 37 &#176;C incubation, slow rotation can be used to improve the distribution of the antibody solution.
<ol>
	<li>Dilute the 5x ligation buffer in high-purity water. Prepare 10 &#181;L per well if using 384-well plates or 20 &#181;L if using coverslips.</li>
	<li>Tap off the PLA probe solution. Wash 1x with TBS-T. Follow with three additional washes, each lasting 5 min, using TBS-T. Wash a final time and tap off the TBS-T before the next step.</li>
	<li>Add Ligase to the 1x Ligation solution at 1:40, immediately before applying it to the samples. Incubate in the pre-heated humidity chamber at 37 &#176;C for&#160;30 min to 1 h.</li>
</ol>
8. Amplification and washing
NOTE: Wait to add the polymerase until immediately prior to addition to the sample. The amplification buffer is light sensitive; protect all solutions containing the buffer from light. Polymerase must be kept in a freezer block (-20 &#176;C). Avoid leaving the cells without a solution for a long period of time. When using 384-well plates for 37 &#176;C incubation, slow rotation can improve the distribution of the antibody solution.
<ol>
	<li>Remove completely all the wash solution before adding the polymerase solution. Avoid leaving the cells without a solution for a long period of time. Dilute the 5x amplification buffer in high-purity water. Prepare 10 &#181;L per well if using 384-well plates or 20 &#181;L if using coverslips.</li>
	<li>Tap off the ligation solution. Wash 1x with TBS-T. Follow with three additional washes, each lasting 5 min, using TBS-T. Wash a final time and tap off the TBS-T before the next step.</li>
	<li>Add the polymerase to the 1x amplification solution at 1:80 dilution, immediately before applying it to the samples. Incubate in the pre-heated humidity chamber at 37 &#176;C for 100 min. 
	NOTE: From this step on, avoid direct light exposition</li>
	<li>Tap off the amplification buffer. Wash 2x with 1x SSC buffer for 10 min each. Wash 2x with 0.01x SSC buffer for 2 min each.</li>
</ol>
9. &#947;-H2AX staining (Immunofluorescence - IF)
NOTE: The labeling of &#947;-H2AX is performed after the PLA is finished.
<ol>
	<li>Primary antibody incubation (IF)
	<ol>
		<li>Dilute the primary antibodies (Rabbit anti yH2AX at 1:100) in blocking solution (alternatively, 3% of Bovine Serum Albumin and Triton X-100 0.1% in PBS solution can be used). Prepare 30 &#181;L of antibody solution per well if using coverslips or 20 &#181;L if using a 384-well plate.</li>
		<li>Tap off the 0.01x SSC solution. Wash 2x with PBS.</li>
		<li>Add the primary antibody solution to each well. Incubate at room temperature for 1 h.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation (IF) 
	NOTE: Make sure the chosen secondary antibodies do not recognize the PLA antibodies or probes. Additionally, the wavelength of the secondary antibody must be different from the PLA detection reagent wavelength. For example: our PLA detection reagent contains the fluorophore 647, and the secondary we used for &#947;-H2AX detection is 488.
	<ol>
		<li>Dilute the secondary antibodies (Donkey anti-Rabbit Alexa Fluor 488) in blocking solution (alternatively, 3% of Bovine Serum Albumin and Triton X - 100 0.1% in PBS solution can be used) at 1:300 dilution. Prepare 30 &#181;L of antibody solution per condition if using coverslips or 20 &#181;L per well if using a 384-well plate. Add Hoechst 33342 dye at 0.6 &#181;g/mL final concentration.</li>
		<li>Tap off the primary antibody solution. Wash 3x with TBS-T.</li>
		<li>Add the secondary antibody solution to each well. Incubate at room temperature for 20 min.</li>
		<li>Wash 5x with TBS-T. Wash 2x with 1x SSC buffer. Wash 2x with 0.01x SSC buffer. 
		NOTE: At this point, the plates can be stored for several weeks at 4 &#176;C (in SSC 0.01X) protected from light. If using coverslips, the slides can be mounted, and after 1 day, the slides can be stored at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
10. Image acquisition
<ol>
	<li>Use a conventional fluorescence microscope and the 63x objective. Determine the time of exposition by comparing negative and positive controls to avoid saturation and dots collapsing. 
	NOTE: The use of confocal microscopes enhances the PLA dots detection. The 20x objective also can be used for increasing the number of nuclei detected.</li>
	<li>Once the exposition time is adjusted, perform all acquisitions of PLA dots with the same parameters. Try to acquire images from different wells or coverslips regions to avoid artifacts due to non-homogeneous incubation.</li>
	<li>Save all the channels with the same name pattern. If the microscope takes photos in the channels separately, save the corresponding images with the same name, adding only PLA or Nucleus at the beginning of the file name to differentiate the channel.</li>
</ol>
11. Image analysis
NOTE: A&#160;macro was created for analysis of dots per nucleus in Image J/FIJI17 and to be used with microscopes where the scale information must be added manually. The pixel size for the objective lens used must be known. Place the images for each channel in different folders (Supplementary Coding File 1, Supplementary Figure 1). 
NOTE: The macro considers fluorescence channels obtained separately. In this case, it is important to save all the images with the same name.
<ol>
	<li>The macro allows the selection of a specific region in the field. Use the option Select a ROI when a region in the image shows artifacts that can be detrimental to analysis. Choose ROI at the beginning of the analysis and use it for all the images. 
	NOTE: This is an optional feature. If not selected, the entire image will be considered in the analysis (Supplementary Figure 2).</li>
	<li>The ROI file should be created prior to the macro usage, as described below. For ROI selection, use a rectangle tool, select the region for analysis, and add it to the ROI manager by clicking Edit &#62; Selection&#62; Add to Manager. Save the ROI by clicking More&#62; Save.</li>
	<li>In the ImageJ 17, open images from different conditions to adjust the parameters for analysis. These parameters must be determined by the user and specified, along with the data, in the macro program (Supplementary Coding File 1)
	<ol>
		<li>Background correction: To remove the background click Process&#62; Subtract Background. Test different rolling ball radius using the preview function). We have used rolling 10 for PLA dots and rolling 100 for nucleus.</li>
		<li>Remove noise by clicking Process &#62; Noise &#62; Despeckle. For nucleus, use the function smooth to improve the filling of the nucleus by clicking Process&#62;Smooth.</li>
		<li>Adjusting threshold: Convert to 8-bit image and adjust the threshold by clicking Image &#62; Type &#62; 8 bit and Image &#62; Adjust &#62;Threshold. Adjust the threshold accordingly to visualize all the nucleus or dots present in the image but avoiding over saturation and structures collapsing. Take notes of the values and test in different images to check if the threshold works for different images.</li>
		<li>Convert to mask&#160;by clicking Process &#62; Binary &#62; Convert to mask. In the case of the nucleus, click Process &#62; Binary &#62; Fill holes followed by Process &#62; Binary &#62; Watershed. In the case of the PLA dots, click Process &#62; Binary &#62; Watershed.</li>
		<li>For counting nuclei, measure the area or length of the different nuclei to estimate the average size. Use an approximate value to analyze particles. Click Analyze &#62; Analyze particles, then select Set size: 15-infinity (these values depend on the size of the nuclei). Check the following options: Show: Mask; Display results; Clear results; Summarize; Add to Manager; Exclude on edges.</li>
		<li>For counting PLA dots, measure the area or the radius of the dots to estimate the average size.&#160;Select Apply Nucleus Mask (apply mask is a function on GDSC plugin, check if it is installed) followed by Analyze &#62; Analyze particles&#160;and set. 0.02-3 (these values depend on the size of the nuclei). Check the following options: Show: Mask; Display results; Clear results; Summarize; Add to Manager; Exclude on edges.</li>
	</ol>
	</li>
	<li>The results show the number of dots per nucleus in each nucleus present in the image. Save this data.</li>
</ol>
12. Visualizing the merged images
<ol>
	<li>Open the channels corresponding to PLA, nucleus, and &#947;-H2AX. Subtract the background and remove noise, as mentioned in steps 11.4.1 and 11.4.2 for all images.</li>
	<li>Adjust brightness and contrast automatically or manually using the same value for all conditions from the same channel.&#160;Select Image &#62; Adjust &#62; Bright/contrast.</li>
	<li>Combine images (merging)by selecting Image &#62; Color &#62; Merge channels. Add scale bar by selecting Analyze &#62; Tools &#62; Scale bar. Save as.TIFF file.</li>
</ol>

Assessing Protein Interaction and Localization in DNA Damage

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The authors declare that they have no competing financial interests.

The data show that the use of PLA concomitantly to a DNA-damaged marker can provide the most information in a DNA damage response profiling, showing the spatial and temporal behavior of the interaction after the insult. PLA is a versatile method that has been used for dimerization identification, organelles contact determination, protein-nucleic acid interaction, and mainly protein-protein interaction10,11,19,<sup cla...

Cellular DNA damage occurs daily because of the spontaneous chemical reactions and is also increased by exogenous factors such as genotoxic agents (radiation and chemicals such as etoposide) and oxidative stress1,2,3. The cells have a complex machinery that corrects a myriad of different types of DNA damage, from removal of bases to replicative fork torsion or interruption to the most deleterious lesion: the DNA double-strand break4,5.
Several proteins that participate in the DDR have already been characterized, and excellent revisions explore the pathways of non-homologous end join (NHEJ) and homologous recombination (HR), the main pathways implied in double-strand breaks (DSB) repair5,6. The phosphorylation of the histone H2A variant, H2AX, at serine 139 (thereafter named y-H2AX) has been used for many years as a sensitive and reliable marker of the double-strand break. The phosphorylation of H2AX is observed in the first minutes following the damage and can persist for hours7.
The advantage of using the immunofluorescence detection of &#947;-H2AX foci is the characterization of the temporal and spatial distribution of the foci, as well as its co-staining with other proteins. Moreover, immunofluorescence does not require lysis, which preserves cellular context information and makes it more informative. Besides, as &#947;-H2AX is formed de novo, it is not abundantly present in the absence of DNA damage, making it a specific marker7.
H2AX is mostly phosphorylated by protein kinase ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), the sensors of DSBs. Ku70/80 (XRCC6 X-ray repair cross-complementing 6/ XRCC5 X-ray repair cross-complementing 5) and DNA-protein kinase catalytic subunit (DNA-PKcs) are responsible for DSB identification and the protection of DNA ends, whereas Artemis carries out end-processing to facilitate ligation by the XRCC4-Ligase IV complex. The phosphorylation of H2AX at sites flanking DSBs acts as a signal for the recruitment of several repair proteins and signaling for cell cycle arrest, death, or survival8.
The analysis of changes in y-H2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response. Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the co-localization of the protein of interest with y-H2AX foci. However, except for the new super-resolution fluorescence methods, concluding the local interaction with DNA damage sites can be tricky. Furthermore, immunofluorescence detection usually depends on many protein molecules at the DNA damage site, making it difficult to identify scarce proteins9.
Here, we show an assay to evaluate the localization of proteins involved in the DDR pathway using y-H2AX as a marker of the damage site. This assay can be used to characterize temporal localization under different insults that cause DNA damage.
The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures10,11and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is like conventional immunofluorescence and allows the simultaneous staining of organelle or cellular structure-specific markers, such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX. The use of PLA allows the identification of protein interaction during DNA damage in a sensitive, specific, and reliable manner that can be used to monitor the interaction during the cell cycle, in response to different stimuli, and at different times after genotoxic stress.
We show here the use of PLA concomitantly to conventional &#947;-H2AX immunofluorescence to localize Nek4-Ku70 interaction after etoposide-induced DNA damage. Nek4, a Nek (NIMA-related kinases) family member, has no clear role in DNA Damage response. In 2012, Nguyen and colleagues showed that Nek4 interacts with Ku70/Ku80 proteins, and in the Nek4 absence, these proteins are not recruited to the DNA damage site, and H2AX phosphorylation is impaired12. The cellular localization of this interaction is unknown so far. We have observed a reduction in Ku70 phosphorylation in cells expressing Nek4 kinase-dead mutant13but whether Nek4 phosphorylates directly Ku70 remains to be elucidated. Considering that Nek4 interaction with Ku70 is increased after etoposide treatment according to immunoprecipitation studies12, we attempt to localize this interaction using PLA and the &#947;-H2AX marker.
Usually, the interaction studies are based on immunoprecipitation assays, but these are in vitro and do not provide information about the location of the interaction. When possible, an immunoprecipitation of fractioned cells (nucleus, mitochondrial, cellular membrane, or cytosol) can be performed, but performing a time course of the interaction is costly and laborious. Immunofluorescence can give information about the cellular localization of the proteins, but proving the interaction can be difficult and depends on the resolution of the microscopes. Here, we show the advantage of using the Proximity Ligand Assay to monitor the interaction of two proteins in a DNA damage context.

<table><tbody><tr><td>Black 384-well plates</td><td>Perkin Elmer Cell carrier plates</td><td></td><td></td></tr><tr><td>Donkey&amp;nbsp; anti- Rabbit Alexa Fluor 488</td><td>Invitrogen</td><td>A21206</td><td>1:300</td></tr><tr><td>Duo link Donkey anti Mouse Minus</td><td>Sigma</td><td>DUO92004</td><td></td></tr><tr><td>Duolink antibody diluent</td><td>Sigma</td><td>DUO82008</td><td></td></tr><tr><td>Duolink blocking solution 1X</td><td>Sigma</td><td>DUO82007</td><td></td></tr><tr><td>Duolink Detection reagent Far red</td><td>Sigma</td><td>DUO92013</td><td></td></tr><tr><td>Duolink Donkey anti goat plus</td><td>Sigma</td><td>DUO92003</td><td></td></tr><tr><td>Duolink Donkey anti rabbit plus</td><td>Sigma</td><td>DUO92002</td><td></td></tr><tr><td>Etoposide</td><td>Sigma</td><td>E1383</td><td></td></tr><tr><td>Goat anti Nek4</td><td>Santa Cruz Biotechnology</td><td>SC-5517</td><td>goat anti Nek4 was used at 1:50 dilution</td></tr><tr><td>Hoechst 33342</td><td>Thermo</td><td>H1399</td><td>0.6 &amp;micro;g/mL</td></tr><tr><td>Leica DMI microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Mouse anti Ku70</td><td>Thermo</td><td>MA5-13110</td><td>mouse anti Ku70 was used at 1:100 dilution</td></tr><tr><td>Mouse anti TOPII&amp;beta;</td><td>Santa Cruz Biotechnology</td><td>SC-365071</td><td>1:25</td></tr><tr><td>Rabbit anti Nek5</td><td>Santa Cruz Biotechnology</td><td>SC-84527</td><td>1:25</td></tr><tr><td>Rabbit anti Y H2AX</td><td>Cell Signalling</td><td>9718S</td><td>1:100 dilution</td></tr><tr><td>U2OS cell line</td><td>ATCC</td><td>HTB-96&amp;nbsp;</td><td></td></tr></tbody></table>

proximity ligand assay to localize proteins in dna damage sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is similar to conventional immunofluorescence and allows the staining of an organelle, cellular structure, or a specific marker such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX simultaneously. The phosphorylation of the S139 at Histone 2A variant, H2AX, then referred to as yH2AX, is widely used as a very sensitive and specific marker of DNA double-strand breaks. Each focus of yH2AX staining corresponds to one break in DNA that occurs a few minutes after the damage. The analysis of changes in yH2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response (DDR). Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the colocalization of the protein of interest with yH2AX foci. However, except for the new super-resolution fluorescence methods, to conclude, the local interaction with DNA damage sites can be a little subjective. Here, we show an assay to evaluate the localization of proteins in the DDR pathway using yH2AX as a marker of the damage site. This assay can be used to characterize the temporal localization under different insults that cause DNA damage.

We have observed Nek4-Ku70 interaction in the absence of etoposide treatment. However, this interaction can occur outside of the nucleus (Figure 1A). The Nek4-Ku70 interaction increases after DNA damage and is concentrated in the nucleus (Figure 1A). In the case of Nek5-Topoisomerase II &#946; (TOPII&#946;) interaction, used in this assay as a positive control based on literature results18, the interaction is greatly increased by etoposid...

Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response

Here, we present a simple and rapid protocol for the detection of protein interaction at DNA damage sites.

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that ...

proximity-ligand-assay-to-localize-proteins-in-dna-damage-sites

Research

JoVE Journal

Biology

411 Views.  University of Campinas. De reactie op DNA-schade is een cellulair systeem dat de stabiliteit en integriteit van het genoom handhaaft. Disfuncties in dit proces veroorzaken verschillende ziekten, zoals kanker, ouderdomsziekten en chronische ontstekingen. Ons doel is om eiwitten te identificeren die samenwerken in dit proces, waardoor alternatieve doelen voor therapeutische integratie kunnen worden geïdentificeerd.Om de deelname van een eiwit aan de DNA-schaderespons te karakt...

Video: Auteur in de schijnwerpers: Proximity-ligandtest combineren met Gamma-H2AX-kleuring om eiwitinteracties in DNA-schaderespons te karakteriseren

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

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 ...

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

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

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

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

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often ...

Cancer Research

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. ...

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 ...

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 ...

Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Source: Wang, X., et al., <a href="https://app.jove.com/v/60982">In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay.</a>&#160;J. Vis. Exp.&#160; (2020)
This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.

Source: Wang, X., et al., In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay.&#160;J. Vis. ...

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

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