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 &#34;home-made PFA&#34;, 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(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-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 (MgCl2)</td>
			<td>Research Products International</td>
			<td>M24000</td>
			<td>Nuclear extraction buffer.</td>
		</tr>
		<tr>
			<td>Piperazine-N,N&#8242;-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.<...

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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 ...

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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

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

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 ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...