Name: visualization of dna repair proteins interaction by immunofluorescence
Uploaded: 2020-06-26T14:45:00
Description: Watch this Scientific Journal Video about Visualization of DNA Repair Proteins Interaction by Immunofluorescence at JoVE.com

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...

<ol>
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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.<...

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 ...

Indirect immunofluorescence makes it possible to detect DNA repair proteins, photos of spatial and temporal recruitment and it helps interrogate Proteinprotein interactions at the site of DNA damage. Following DNA damage, DNA repair proteins are recruited to the DNA insult while their concentration increases locally, and they form groups called foci that can be visualized by indirect immunofluorescence on fixed samples. This technique can be used to detect protein foci, as well as to quantify colocalization of the present in cells.This can help explain the sequence of events required for complex formation and for DNA repair. particular proteinprotein interactions can be entailed from such experiments. Demonstrating the procedure will be Barbara De La Pena, a very talented Postdoc Photo from my laboratory Begin by growing 40, 000 HeLa cells in each well over 12 well plate with an 18 millimeter round glass coverslip to 80%confluency.After exposing the cells to four gray gamma irradiation, wash them twice with one milliliter of PBS. Then remove the PBS completely and add 200 microliters of NDB to each well. Incubate the cells for two minutes at room temperature then remove the NDB.Keep in mind that incubation time can vary depending on the cell line, but generally should not exceed two minutes. Wash the cells with one milliliter of PBS. Then remove the PBS completely and add 200 microliters of 4%PFA to each well for cell fixation.Incubate the cells for 10 minutes at four degrees Celsius. Then remove the PFA and add one milliliter of PBS to each well. Remove PBS completely and then add 200 microliters of blocking solution to each well and incubate the cells for two hours at room temperature or 16 to 18 hours at four degrees Celsius.dilute the primary antibody and dilution buffer and vortex it until well mixed in a humidity box and here a piece of parafilm and add 10 microliters of primary antibody in a single drop. Align one edge of the coverslip from the well with the drop and slowly lower it onto the parafilm spreading the liquid. incubate the coverslip for two hours at room temperature.After the incubation wash the cover slips three times in PBS for one minute per wash. dilute the secondary antibody and dilution buffer and vortex it until well mixed apply 10 microliters of secondary antibody to each coverslip as previously described and incubate it for two hours at room temperature protected from light. Wash the cover slips three times with PBS and once with water for one minute per wash.Then mount them onto glass slides with a glycerol based mounting media containing DAPI seal cover slips with transparent nail polish and let them dry for 20 minutes. Place a drop of immersion oil onto the 60 x objective lens and use DAPI to locate the nuclei through the eyepiece for XYZ image acquisition, open the acquisition software and set the scanner type as galvano the scanner type as roundtrip and 512 by 512 image size. In the PMT panel set mode to VBM average to frame and sequential scan to line.Next set the dye and heat detectors set channel one to DAPI and SD one, channel two to alexafluor 488 and HSD three, and channel three to alexafluor at 647 and HSD four select on to Z.To adjust the live image, press the live button on the live window and adjust the focus. Then use the PMT tool window to set laser intensity sensitivity, gain and offset For z stacks, select start to end and 15 slices. Select the folder to save images and press the LSM Start button to start acquiring.When finished, press the series done button to complete the image acquisition. Open the analysis software then press the batch tool window and select the images to analyze. Go to the analysis tool window and select projection, which will display the maximum intensity projection from 15 slices.Under the input output setting, select the created batch and output folder. Press process for images to be processed and export the images as TIFF files. Perform nuclear quantification with cellprofiler according to manuscript directions.Cells not treated with the radiation exhibit very few gamma H2AX expo sign. In the absence of essential DNA repair proteins, gamma H2AX accumulation can be observed at DNA breaks. Accumulation of unrepaired breaks can lead cells to become pre hypotonic indicated by solid gamma H2AX nuclei.Following irradiation, nuclei exhibit a large number of double stranded breaks to which gamma H2AX localize is extremely rapidly. While few if any foci are observed in the absence of a radiation the number of foci was quantified at one two four and 16 hours post a radiation and for the NOA radiation control. Depending on the biological question raised and the type of data required, different plotting options should be considered Colocalization can be investigated by multiplexing primary antibodies raised in different animal species and using secondary antibodies, are labeled with distinct fluorophores.Superposition of green and red, gives rise to yellow hotspots, the two proteins of interest are present in the same pixels. Quantitative analysis of colocalization can be achieved by an object based approach or by a statistical approach that performs intensity correlation coefficient based analyses. A combination of primary antibodies raised in different animal species, can be used in this protocol.Make sure that the antibodies are compatible and will not interfere with each other. You need to appropriately set the secondary antibody to be used against each of the primary antibodies and consider a particular spectral overlap when selecting the fewer force to be used. Colocalisation or proteins indicate possible direct interactions.This can be verified by granular precipitation in cells, or by direct put down using purified proteins in vitro.

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