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 ...

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