This work was supported by a grant from the San Antonio Area Foundation. The Mays Cancer Center is supported by NCI cancer center support core grant P30 CA054174. We would like to thank Stephen Holloway for his help sourcing the reagents, and the Sung laboratory for providing space and microscopy capacity.

1. HeLa cell culture
<ol>
	<li>Pre-treat round glass coverslips with 1 M HCl at 50 &#176;C for 16-18 hours. Rinse with ddH2O and store in 100% EtOH.</li>
	<li>Prepare cell culture medium: add 10% (v/v) FBS to DMEM medium.</li>
	<li>Grow 4.0 x 104 cells/well in sterile 12-well plate with an 18 mm round glass coverslip at 37 &#176;C and 5% CO2 in a humidified incubator. Grow cells to 80% confluency (approximately 24 hours).</li>
</ol>
2. Cell treatment with radiation (IR)
<ol>
	<li>To induce the formation of double-strand breaks, expose cells to 4 Gy &#947;-irradiation (control: No irradiation, t=0). In the Gamma Cell -40, this corresponds to 4.54 min, at 0.815 Gy per min.</li>
	<li>Incubate cells at 37 &#176;C and 5% CO2 in a humidified incubator for the appropriate time-length (time points chosen here t=1, 2, 4, 16 h).</li>
</ol>
3. Nuclear extraction and cell fixation
<ol>
	<li>Prepare stock solutions: 0.2 M PIPES (pH 6.8), 5 M NaCl, 2 M sucrose, 1 M MgCl2, 0.1 M EGTA (pH 8.0).</li>
	<li>Prepare nuclear extraction buffer (NEB): dissolve 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0) and 0.5% (v/v) Triton X-100 in ddH2O. Mix until dissolved completely.</li>
	<li>Prepare 4% (v/v) paraformaldehyde (PFA): dissolve 10 mL of 16% PFA aqueous solution in 30 mL PBS. Mix until dissolved completely.</li>
	<li>At appropriate time point (t=0, 1, 2, 4, 16 h), wash cells twice with 1 mL of PBS. Remove PBS completely.</li>
	<li>Add 200 &#956;L of NEB to each well for cell nuclei extraction (cytoplasm is degraded, only nucleus remains) (Figure 2). Incubate for 2 minutes at room temperature and remove completely. 
	NOTE: Do not exceed 2 minutes.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61447/61447fig02.jpg" /> 
Figure 2: Nuclear extraction. 
Representative images of cells prior to (left) and post (right) nuclear extraction. Cytoplasm should be digested but the nuclear structure should remain intact post extraction (right). (A) 20x magnification; scale bar = 20 &#956;m and (B) 40x magnification; scale bar = 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/61447/61447fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Wash cells with 1 mL of PBS. Remove PBS completely. Add PBS carefully, cells are very fragile at this step.</li>
	<li>Add 200 &#956;L of 4% (v/v) PFA to each well for cell fixation. Incubate for 10 minutes at 4 &#176;C. Remove PFA completely.</li>
	<li>Add 1 mL of PBS. 
	NOTE: Cells can be stored in PBS at 4 &#176;C.</li>
</ol>
4. Immunofluorescence staining
<ol>
	<li>Prepare blocking solution: dissolve 5% BSA (w/v) in PBS and add 0.3% (v/v) Triton X-100. Mix until completely dissolved.</li>
	<li>Prepare dilution buffer: dissolve 1% BSA (w/v) in PBS and add 0.3% (v/v) Triton X-100. Mix until completely dissolved.</li>
	<li>For blocking, add 200 &#956;L of blocking solution to each well. Incubate for 2 hours at room temperature or 16-18 hours at 4 &#176;C. 
	NOTE: If goat antibody will be used, add 5% goat serum to blocking solution.</li>
	<li>Dilute primary antibody in dilution buffer (1:500;&#160;see Table 2 for antibodies list) and vortex until well mixed. 
	NOTE: If goat antibody is used, add 1% goat serum to dilution buffer.</li>
	<li>In a humidity/incubation box, adhere a piece of parafilm. Add 10 &#956;L of primary antibody (in a single drop). Align one edge of the coverslip with the drop and slowly lower onto the parafilm, for the liquid to spread throughout (avoid bubbles if possible). Incubate for 2 hours at room temperature.</li>
	<li>Wash coverslips three times in PBS for 1 minute.</li>
	<li>Dilute secondary antibody in dilution buffer (final concentration: 2 &#956;g/mL) and vortex until well mixed.</li>
	<li>Apply 10 &#956;L of secondary antibody as described for the primary antibodies. Incubate for 2 hours at room temperature. 
	NOTE: Protect from light.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Company</td>
			<td>Reference</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>53BP1</td>
			<td>Cell Signaling</td>
			<td>4937</td>
			<td>Rabbit</td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG H&#38;L (Alexa Fluor 647)</td>
			<td>Abcam</td>
			<td>ab150103</td>
			<td>Donkey</td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H2A.X (Ser139), clone JBW301</td>
			<td>Millipore</td>
			<td>05-636</td>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150081</td>
			<td>Goat</td>
		</tr>
	</tbody>
</table>
Table 2: Antibodies used. List of antibodies used in this study.
<ol start="9">
	<li>Wash coverslips three times in PBS for 1 minute.</li>
	<li>Wash coverslips with H2O for 1 minute.</li>
	<li>Counterstain DNA with DAPI: apply 10 &#956;L of 300 nM DAPI (as described for antibodies), incubate for 30 minutes at room temperature and then mount onto glass slide with a glycerol based mounting media. Alternatively, add one drop (10 &#956;L) of commercial antifade mounting media containing DAPI onto a slide and apply a coverslip. Gently press the coverslip and remove excess fluid around it with a paper towel.</li>
	<li>Seal coverslips with transparent nail polish and let them dry for 20 minutes.</li>
	<li>Store slides at 4 &#176;C.</li>
</ol>
5. Image acquisition
<ol>
	<li>Place a drop of immersion oil onto the 60x objective lens. Use DAPI to locate the nuclei through eye piece.
	<ol>
		<li>For XYZ image acquisition, open acquisition software and select parameters: Scanner type: Galvano; Scanner mode: Roundtrip; Image size: 512&#215;512; PMT mode: VBF; PMT average: frame (4 times); PMT sequential scan: line.</li>
		<li>Select the dye and the detectors: 
		Channel (CH1), Dye (DAPI), Detector (SD1) 
		Channel (CH2), Dye (Alexa Fluor 488), Detector (HSD3) 
		Channel (CH3), Dye (Alexa Fluor 647), Detector (HSD4)</li>
		<li>Select ON in &#8220;Z&#8221;.</li>
	</ol>
	</li>
	<li>Adjust the live image. Press the Live button on the Live window.
	<ol>
		<li>Adjust the focus and set laser intensity (%), sensitivity (HV), gain and offset on &#8220;PMT&#8221; tool window.
		<ol>
			<li>Adjust the laser intensity (%): for brightness and bleaching. The higher the laser intensity, the stronger the signal, but the specimen will photobleach.</li>
			<li>Adjust the sensitivity (HV): noise level. The higher the HV, the stronger the signal, but image will be noisy if too high. 
			NOTE: Always keep voltage constant.</li>
			<li>Adjust the offset: background level.</li>
		</ol>
		</li>
		<li>Select Start/End (15 slices), for Z stacks.</li>
	</ol>
	</li>
	<li>Start the acquisition.
	<ol>
		<li>Select the folder to save images. Press the LSM Start button to start acquiring the image. Press the Series Done button to complete the image acquisition.</li>
	</ol>
	</li>
</ol>
6. Data analysis
<ol>
	<li>For image analysis, open the analysis software.
	<ol>
		<li>Press the Batch tool window,&#160;select the images to analyze and select output folder.</li>
		<li>Press the Analysis tool window and select Projection (will display the maximum intensity projection from 15 slices).</li>
		<li>Under Input/Output setting, select the batch created.</li>
		<li>Press Process for images to be processed.</li>
		<li>Export images as .tiff files.</li>
	</ol>
	</li>
	<li>For nuclear foci quantification, open CellProfiler.
	<ol>
		<li>Open the &#947;H2AX and 53BP1 foci quantification pipeline (see Supplemental information).</li>
		<li>Graph data using a table software.</li>
	</ol>
	</li>
	<li>For co-localization analysis, open CellProfiler.
	<ol>
		<li>Open the Colocalization pipeline (see Supplemental information). A spreadsheet file will be created and saved in the preferred location. However, graphs themselves will not be automatically saved. It is suggested to take a snapshot of the windows to keep for record of the results.</li>
		<li>Graph data using a table software.</li>
	</ol>
	</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+Radiation+and+Complex+DNA+Damage:+From+Prediction+to+Detection+Challenges+and+Biological+Significance.">Ionizing Radiation and Complex DNA Damage: From Prediction to Detection Challenges and Biological Significance.</a> Cancers (Basel). 11 (11), (2019).</li><li>Nikitaki, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+complex+DNA+damage+induction+and+repair+in+human+cellular+systems+after+exposure+to+ionizing+radiations+of+varying+linear+energy+transfer+(LET).">Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET).</a> Free Radical Research. 50, 64-78 (2016).</li><li>Redon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H2A+variants+H2AX+and+H2AZ.">Histone H2A variants H2AX and H2AZ.</a> Current Opinion in Genetics &amp; Development. 12 (2), 162-169 (2002).</li><li>Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+the+histone+guardian+of+the+genome.">H2AX: the histone guardian of the genome.</a> DNA Repair. 3 (8-9), 959-967 (2004).</li><li>Paull, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+histone+H2AX+in+recruitment+of+repair+factors+to+nuclear+foci+after+DNA+damage.">A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.</a> Current Biology. 10 (15), 886-895 (2000).</li><li>Sy, S. M. H., Huen, M. S. Y., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PALB2+is+an+integral+component+of+the+BRCA+complex+required+for+homologous+recombination+repair.">PALB2 is an integral component of the BRCA complex required for homologous recombination repair.</a> Proceedings of the National Academy of Sciences. 106 (17), 7155-7160 (2009).</li><li>Buisson, R., Masson, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PALB2+self-interaction+controls+homologous+recombination.">PALB2 self-interaction controls homologous recombination.</a> Nucleic Acids Research. 40 (20), 10312-10323 (2012).</li><li>Belotserkovskaya, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PALB2+chromatin+recruitment+restores+homologous+recombination+in+BRCA1-deficient+cells+depleted+of+53BP1.">PALB2 chromatin recruitment restores homologous recombination in BRCA1-deficient cells depleted of 53BP1.</a> Nature Communications. 11 (1), 819 (2020).</li><li>Betts, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+noncoding+RNAs+CUPID1+and+CUPID2+mediate+breast+cancer+risk+at+11q13+by+modulating+the+response+to+DNA+damage.">Long noncoding RNAs CUPID1 and CUPID2 mediate breast cancer risk at 11q13 by modulating the response to DNA damage.</a> American Journal of Human Genetics. 101 (2), 255-266 (2017).</li><li>Dray, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+enhancement+of+the+meiotic+DMC1+recombinase+by+RAD51+associated+protein+1+(RAD51AP1).">Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1).</a> Proceedings of the National Academy of Sciences. 108 (9), 3560-3565 (2011).</li></ol>

The authors have nothing to disclose.

Analysis of the timing and efficiency of DNA damage repair by microscopy has proven essential to understand how the DNA repair machinery functions, in normal cells and in human pathologies such as cancer.
The development of specific antibodies that allow detection of activated proteins in their phosphorylated version (such as &#947;H2AX, pRPA, pRAD50 and others10,23,39,43<...

Human cells are constantly exposed to a variety of DNA damaging agents of various origins. Exogenous sources mostly consist of exposure to radiations, chemicals (including chemotherapeutic agents and some antibiotics), and viruses, while the main endogenous sources include errors in DNA replication and oxidative stress. The direct effects of genotoxic exposure can range from a modified base to a potentially lethal DNA double-strand break (DSB), depending on the stress and the exposure dose. Ultimately, unrepaired or mis-repaired DNA damage can lead to the accumulation of mutations, genomic rearrangements, genome instability and eventually lead to carcinogenesis1. Mammalian cells have evolved complex pathways to recognize specific types of DNA damage2,3 and repair them in a timely fashion, synchronized with the cell cycle progression.
Ionizing radiation (IR) damages the DNA double helix and creates double-strand breaks (DSBs), one of the most deleterious forms of DNA damage. The MRN (MRE11, RAD50, NBS1) complex functions as a sensor of DNA ends and activates the protein kinase ataxia telangiectasia mutated (ATM)4,5. Following the initial activation of ATM by DNA ends, ATM triggers a cascade of DDR events at the site of the break, initiating with a key event, the phosphorylation of the histone variant H2AX6. H2AX phosphorylation on residue S139 activates it into &#947;H2AX, spanning regions up to megabases around the DNA lesion6,7,8,9. This event increases DNA accessibility, leading to the recruitment and accumulation of other DNA repair proteins7. Because &#947;H2AX is abundantly and specifically induced surrounding DSBs, it can be readily visualized using specific antibodies, and is commonly used as a surrogate marker for DSBs in the DNA repair field. Once the break is signaled, cells activate their DNA repair pathways and process the DNA damage. The protein MDC1 (mediator of DNA damage checkpoint protein 1) directly binds &#947;H2AX10, interacts with ATM11 and also with NBS112,13. It contributes to increasing the concentration of MRN complex at the DSB and initiating a positive ATM feedback loop. &#947;H2AX is rapidly removed once the break is repaired, consequently, allowing the monitoring of DSB clearance. Followed by microscopy, the decrease in &#947;H2AX over time provides an indirect measurement of residual breaks and DNA repair efficiency.
Eukaryotic cells can repair DSBs by several pathways, the two main ones being non-homologous end-joining (NHEJ) and homologous recombination (HR) (Figure 1). NHEJ essentially ligates DNA double-strand ends without the use of extended homology and operates throughout the cell cycle14,15. HR becomes predominant during S and G2 phases, and is otherwise repressed, since it requires a sister chromatid as a homologous template for repair14,16. Pathway choice between NHEJ and HR not only depends on the physical proximity of the sister chromatid, but also on the extend of DNA end resection17, which inhibits NHEJ.
Homology-dependent DSB repair initiates by nucleolytic degradation of the 5&#8217; strand from the break ends to generate 3&#8217; single-strand DNA (ssDNA) tails, a process referred to as 5&#8217;-3&#8217; resection. The MRN complex initiates DNA end resection and further resection is processed in combination with BLM/EXO1 (Bloom syndrome protein/exonuclease 1) or BLM/DNA2 (DNA replication ATP-dependent helicase/nuclease)18,19,20,21,22. DNA end resection is enhanced by CtIP (CtBP-interacting protein) through its direct interaction with MRN complex23 and recruitment of BRCA1 (breast cancer type 1 susceptibility protein)24,25. Replication protein A (RPA) promptly binds to the ssDNA exposed and is then displaced by the recombinase protein RAD51 to form a nucleoprotein filament that catalyzes homologous search and strand invasion26,27,28.
The initiation of resection is a critical step for repair pathway choice. Once resection has initiated, the DNA ends become poor substrates for binding by Ku70/Ku80 heterodimer (component of NHEJ pathway) and cells are committed to HR17,29,30. The Ku70/Ku80 heterodimer binds to DSB ends, recruiting DNA-PKcs and p53 Binding Protein 1 (53BP1)29,30. 53BP1 acts as a barrier to resection in G1, thus blocking HR while promoting NHEJ31,32, but it is removed in a BRCA1-dependent manner in S phase, consequently allowing resection to occur33,34. Therefore, 53BP1 and BRCA1 play opposing roles in DSB repair, with 53BP1 being a NHEJ facilitator whilst BRCA1 acts enabling breaks to repair through HR.
In the laboratory, DSB formation can be induced by ionizing radiation (IR). While this example utilizes a high dose of 4 Gy, 1 Gy and 2 Gy also create a significant amount of DSBs, suitable for the analysis of foci formation by abundant proteins. It is important to note that the type and dose of radiation used can lead to different lesions in the DNA and in the cell: while IR induces DSBs, it can also cause single strand breaks or base modification (see35,36 for a reference on irradiation linear energy transfer (LET) and type of DNA damage). To determine the kinetics of ionizing radiation-induced foci (IRIF) formation and their clearance, which indicate repair of the damage and reversal of the activated DDR8,9,37,38, foci formation can be monitored at different time points after ionizing radiation. Timing of activation and clearance of all major DNA damage proteins is known39, and many are investigated as surrogate markers of key events. For example, pRPA, which possesses high affinity for ssDNA is used as a surrogate of the break resection, MRN proteins (MRE11, RAD50, NBS1) and exonucleases can be used to assess resection efficiency too. While RAD51, BRCA1, BRCA2 (breast cancer type 2 susceptibility protein), and PALB2 (partner and localizer of BRCA2) can be monitored to evaluate HR efficiency, the presence of the Ku proteins or 53BP1, are used as markers of NHEJ (Figure 1).
As proteins of the DNA repair machinery recruit each other to the break and assemble in super-complexes, DNA-protein and protein-protein interactions can be inferred by following their individual localization over time and analyzing co-localization of proteins, as visualized by overlapping signals in cell40,41,42. In cell lines, the introduction of point mutations or deletion in specific DNA repair genes either through genome editing, or by overexpression of plasmid-based mutants, allows investigation of specific residues and their possible role in recognition of DNA damage (e.g., co-localization with &#947;H2AX) or complex assembly (co-localization with another, or several, proteins), as well as their impact on DNA repair. Here, we use indirect immunofluorescence as a mean to investigate the formation and resolution of DSBs by following &#947;H2AX foci over time. We also present one example of foci formation and co-localization analysis by a major player in DSB repair: p53 Binding Protein 1 (53BP1)32. As mentioned earlier, 53BP1 is considered central to DNA repair pathway choice. Following 53BP1 accumulation and its co-localization with &#947;H2AX provides precious information on cell cycle phase, DNA damage accumulation, and pathway used to repair DSBs. The purpose of indirect immunolocalization is to assess the efficiency of DNA damage repair in cell lines, following IR like in this study, or after exposure to various stresses in cell, from DNA crosslinking to blockage of the replication fork (a list of DNA damaging agents is provided in Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61447/61447fig1v2.jpg" /> 
Figure 1: DNA double strand breaks (DSB) repair pathways. 
DSB repair involves two major pathways: Homologous Recombination (HR, left) and Non-Homologous End-Joining (NHEJ, right). Following the break, proteins get activated to mark the break (&#947;H2AX),&#160;participate in end resection (MRN), coat the resected ssDNA (pRPA), promote recombination (BRCA1, PALB2, BRCA2, RAD51) or limit resection and promote NHEJ (53BP1). Other proteins participate in damage repair, but proteins listed are routinely followed by indirect immunofluorescence. <a href="https://www.jove.com/files/ftp_upload/61447/61447fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DNA damaging agent</td>
			<td>Mechanism of action</td>
			<td>Recommended dose</td>
		</tr>
		<tr>
			<td>&#947;-rays/X-rays</td>
			<td>Radiation 
			Formation of double-stranded breaks with some uncontrolled cellular effects</td>
			<td>1-4 Gy</td>
		</tr>
		<tr>
			<td>36Ar ions</td>
			<td>Radiation 
			Formation of double-stranded breaks</td>
			<td>270&#8201;keV/&#956;m</td>
		</tr>
		<tr>
			<td>&#945;-particles</td>
			<td>Radiation 
			Formation of double-stranded breaks</td>
			<td>116&#8201;keV/&#956;m</td>
		</tr>
		<tr>
			<td>Bleomycin</td>
			<td>Inhibitor of DNA synthesis</td>
			<td>0.4-2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Camptothecin</td>
			<td>Inhibitor of topoisomerase I</td>
			<td>10-200 nM</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Alkylating agent 
			(inducing intrastrand crosslinks)</td>
			<td>0.25-2 &#956;M</td>
		</tr>
		<tr>
			<td>Doxorubicin</td>
			<td>Intercalating agent 
			Inhibitor of topoisomerase II</td>
			<td>10-200 nM</td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Inhibitor of topoisomerase II</td>
			<td>10 &#956;M</td>
		</tr>
		<tr>
			<td>Hydroxyurea</td>
			<td>Inhibitor of DNA synthesis 
			(by ribonucleotide reductase)</td>
			<td>10-200 &#956;M</td>
		</tr>
		<tr>
			<td>Methyl methanesulfonate</td>
			<td>Alkylating agent</td>
			<td>0.25-2 mM</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Alkylating agent</td>
			<td>0.25-2 &#956;M</td>
		</tr>
		<tr>
			<td>Ultraviolet (UV) light</td>
			<td>Formation of thymidine dimers 
			(generating distortion of DNA chain)</td>
			<td>50-100 mJ/cm2</td>
		</tr>
	</tbody>
</table>
Table 1: Genotoxic agents. Examples of DNA damaging agents, their mechanism of action and the damage induced based on suggested working concentration.

<table><tbody><tr><td>16% (v/v) paraformaldehyde (PFA) aqueous solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Microscopy quality of the PFA ensures best images. If using &quot;home-made PFA&quot;, filter prior to use.</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3059</td><td>Heat-shock fraction is recommended, to avoid precipitation/background.</td></tr><tr><td>Coverglass #1, 18 mm round (coverslips)</td><td>Neuvitro</td><td>NC0308920</td><td>Coverslips need to be cleaned and sterilized prior using, with HCl or ethanol.</td></tr><tr><td>DMEM, High Glucose [(+) 4.5 g/L D-Glucose, (+) L-Glutamine, (-) Sodium Pyruvate]</td><td>Gibco</td><td>11965118</td><td>Adjust the growing media to the needs of cell line used.</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190144</td><td>PBS for tissue culture.</td></tr><tr><td>Ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid (EGTA)</td><td>Research Products International</td><td>E57060</td><td>Nuclear extraction buffer.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>104370028</td><td>The quality of FBS can be assessed by testing gH2AX foci formation. If traces of genotoxic endotoxin are present in the batch, gH2AX will be positive in the absence of stress.</td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Research Products International</td><td>M24000</td><td>Nuclear extraction buffer.</td></tr><tr><td>Piperazine-N,N&amp;prime;-bis(2-ethanesulfonic acid) (PIPES)</td><td>Research Products International</td><td>P40150</td><td>Nuclear extraction buffer.</td></tr><tr><td>SlowFade Diamond Antifade Mountant with DAPI</td><td>Invitrogen</td><td>S36973</td><td>300 nM DAPI with VECTASHIELD Antifade Mounting Medium can be used instead.</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Research Products International</td><td>S23020</td><td>Nuclear extraction buffer.</td></tr><tr><td>Sucrose</td><td>Research Products International</td><td>S24060</td><td>Nuclear extraction buffer.</td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisherbrand</td><td>1255015</td><td>Polysine Slides can be used instead.</td></tr><tr><td>TC-Treated Multiple Well Plates, size 12 wells</td><td>Costar</td><td>3513</td><td>Seeding on coverslips is done in 12-wells plate.</td></tr><tr><td>Triton X-100</td><td>AmericanBio</td><td>AB02025</td><td>Nuclear extraction buffer.</td></tr><tr><td>TrypLE Express Enzyme (1X), No Phenol Red</td><td>Gibco</td><td>12604021</td><td>Trypsin-EDTA can be used instead.</td></tr><tr><td>Trypsin-EDTA (0.5%), No Phenol Red</td><td>Gibco</td><td>15400054</td><td>TrypLE can be used instead.</td></tr></tbody></table>

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.

On day 2, or 24 h post seeding cells on coverslips, cells have undergone one division and are 80% confluent. Specific knock downs or mutations in DNA repair proteins can increase doubling time, or sensitize cells to genotoxic treatment, and seeding density as well as treatment doses should be adjusted accordingly. To determine best working conditions, timing of the DNA damage response can be established by Western blotting of &#947;H2AX over time, and sensitivity to irradiation can be identified by colony forming assay.<...

Watch this Scientific Journal Video about Visualization of DNA Repair Proteins Interaction by Immunofluorescence at JoVE.com

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

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 ...

visualization-of-dna-repair-proteins-interaction-by-immunofluorescence

Research

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Biology

10.0K Views.  University of Texas Health Science Center at San Antonio. 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.

Video: Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Cancer Research

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

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Immunology and Infection

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

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 ...

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 ...

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Source: de la Pe&#241;a Avalos, B., et al., <a href="https://app.jove.com/v/61447">Visualization of DNA Repair Proteins Interaction by Immunofluorescence.</a> J. Vis. Exp. (2020).
In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins &#947;H2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

Source: de la Pe&#241;a Avalos, B., et al., Visualization of DNA Repair Proteins Interaction by Immunofluorescence. J. Vis. Exp. (2020).
In this video, we ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

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Chapters in this video

Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

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

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

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

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

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions