Research in the Guikema laboratory is funded by the Innovational Research Incentives Scheme from The Netherlands Organization for Scientific Research (VIDI grant 016126355) and the 'Stichting Kinderen Kankervrij' KiKA (project 252).

1. Treatment of Cells and DNA Damage Induction
<ol>
	<li>Culture the human BCR-ABL+ B-cell acute lymphoblastic cell lines BV173 or SUP-B15 in IMDM supplemented with 20% FCS, 50 &#956;M &#946;-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin and 100 &#956;g/mL streptomycin at 37 &#176;C in an atmosphere of 5% CO2. Count cells and plate at 2 x 106 cell/mL in 6-wells plates, at 5 mL/well. 
	NOTE: Optionally, suspension cell lines of various origin can used.</li>
	<li>To arrest the BCR-ABL+ B-ALL cells in the G1-phase of the cell-cycle, add 5 &#956;M of imatinib methanesulfonate (STI571), and incubate overnight.
	<ol>
		<li>Optionally, omit this step when studying protein-protein interactions throughout the cell-cycle or in BCR-ABL-negative cell types.</li>
	</ol>
	</li>
	<li>Induce DNA damage by adding 50 ng/mL neocarzinostatin (NCS) to the culture, and incubate for 2 h.
	<ol>
		<li>Alternatively, induce DNA damage by irradiating the cells using a radioactive source [137Cs] at 0.5 Gy/min, at a dose of 5 Gy. After irradiation, let the cell recover for 2 h at 37 &#176;C in an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Optionally, to assess the involvement of ATM kinase activity in response to DNA damage, add 5 &#956;M of the ATM kinase inhibitor KU55933 prior to addition of NCS or the irradiation treatment.</li>
	<li>Prepare 1x PBS + 10% BSA by layering 5 g of fraction-V BSA on top of 50 mL of 1x PBS in a beaker or Erlenmeyer flask, and let sit at RT until BSA is dissolved. Do not shake or stir as this will cause extensive foaming. Slowly filter through a 0.22 &#956;m filter and keep on ice.</li>
	<li>Harvest cells and wash twice with ice-cold 1x PBS. Count the cells and resuspend at 2 x 106/mL in 1x PBS + 10% BSA. Keep cells on ice.</li>
</ol>
2. Cytocentrifugation, Fixation and Permeabilization
<ol>
	<li>Prepare the 4% paraformaldehyde solution in 1x PBS freshly. Add 2 g of PFA to 50 mL of 1x PBS and incubate in a 55 &#176;C shaking water bath until the solution is clear. Filter the solution through a 0.22 &#956;m filter and store at RT until use. 
	NOTE: Optionally, 4% PFA fixative can be stored frozen at -20 &#176;C. After freezing, thaw only once for use. Otherwise, the 4% PFA solution in PBS can be purchased directly.</li>
	<li>Carefully polish 76 mm x 26 mm microscopy slides with lint-free tissue and degrease them by placing in 96% ethanol in a Coplin jar for 5 min. Let the microscopy slides air-dry for 10 min.</li>
	<li>Assemble microscopy slides into the cytospin/cytology funnels with an area of 6 mm. Pre-coat slides by pipetting 50 &#956;L of 1x PBS + 10% BSA into each funnel and centrifuge for 1 min at 500 rpm.</li>
	<li>Add 100 &#956;L of the cell-suspension (equaling 0.2 x 106 cells) to each funnel and centrifuge for 5 min at 500 rpm. For each antibody combination, at least 4 preparations are required (dual-antibody experiment; two single-antibody controls; no antibodies control).</li>
	<li>Carefully disassemble the funnels, and make sure not to touch the spots. Proceed to sample fixation immediately.
	<ol>
		<li>Optionally, let slides air-dry for at least 30 min. After air-drying, wrap slides individually in aluminum foil and store at -20 &#176;C until use. When using frozen preparations, make sure to thaw the slides wrapped in a paper towel to prevent overt condensation onto the slides.</li>
	</ol>
	</li>
	<li>Draw a circle (approximate diameter 15-20 mm) around the spot using a PAP pen liquid blocker (5 mm tip).</li>
	<li>Add 50 &#956;L of 4% PFA fixation solution to each spot and incubate for 30 min at RT.</li>
	<li>Wash the cells 3 times for 5 min with 1x PBS in a Coplin jar at RT with gentle agitation.</li>
	<li>Dry the slides around the spots with a lint-free tissue and remove as much liquid from the spot as possible using a pipet without touching the spot, or by tilting the slide. Add 50 &#956;L of 0.25% Triton-X in 1x PBS to each spot and incubate for 10 min at RT without agitation.</li>
	<li>Wash the slides 3 times for 5 min in ~50-60 mL of 0.05% Tween-20 in TBS (TBS-T) in a Coplin jar at RT with gentle agitation. 10 slides can be processed simultaneously in this fashion.</li>
</ol>
3. Blocking and Primary Antibody Incubation
<ol>
	<li>Tap off TBS-T and dry the slides around the spots with a lint-free tissue. Remove as much liquid from the spot as possible using a pipet without touching the spot, or by tilting the slide. Shortly vortex the blocking solution and add one drop (~ 40 &#956;L) to each spot. Incubate for 1 h at 37 &#176;C in a pre-warmed humidified chamber.</li>
	<li>Prepare the antibody cocktail by mixing the goat-anti-ATM at 1:1,000 and the rabbit-anti-phospho-Ser15-p53 at 1:100 in commercial antibody diluent, vortex antibody diluent before use. For control experiments, prepare antibody dilutions containing only the goat-anti-ATM and only the rabbit-anti-phospho-Ser15-p53 antibody (single-antibody controls).</li>
	<li>Tap off the blocking solution but take care not to let the cells dry prior to adding the antibody dilutions. Add 30 &#956;L of each antibody cocktail dilution and incubate in a humidified chamber overnight at 4 &#176;C.</li>
	<li>Prepare in situ Wash Buffer A and Wash Buffer B by dissolving the content of one pouch of Buffer A and Buffer B mix in a final volume of 1,000 mL of water each.
	<ol>
		<li>Alternatively, prepare wash buffer A by dissolving 8.8 g of NaCl, 1.2 g of Tris-base and 0.5 mL of Tween-20 in 800 mL water. Adjust pH to 7.4 using HCl and add to a final volume of 1,000 mL.</li>
		<li>Alternatively, prepare Wash Buffer B by dissolving 5.84 g of NaCl, 4.24 of Tris-base and 26.0 g of Tris-HCl in 500 mL of water. Adjust pH to 7.5 using HCl. Add water to a final volume of 1,000 mL.</li>
		<li>Filter both solutions through a 0.22 &#956;m filter.</li>
	</ol>
	</li>
	<li>Wash the slides 2 times for 5 min with 1x in situ wash buffer A in a Coplin jar at RT with gentle agitation.</li>
</ol>
4. PLA Probes, Ligation and Amplification
<ol>
	<li>Prepare PLA probes by diluting the anti-goat PLUS and anti-rabbit MINUS both 1:5 in the commercial antibody diluent. Tap off 1x in situ wash buffer A from the slides and add 30 &#956;L of the PLA probe-mixture to each spot. Incubate 1 h at 37 &#176;C in a pre-warmed humidified chamber. 
	NOTE: Any combination of secondary antibodies PLUS and MINUS PLA probes can be used (anti-mouse, anti-rabbit, anti-goat), as long as the applied primary antibodies are raised in different species, and the combination of PLA probes should always contain a PLUS probe and a MINUS probe.</li>
	<li>Wash slides 2 times for 5 min with ~50-60 mL 1x in situ wash buffer A in a Coplin jar at RT with gentle agitation.</li>
	<li>Prepare the Ligation-Ligase solution on ice by adding 1 &#956;L of Ligase to 39 &#956;L of Ligation solution and add 40 &#956;L of Ligation-Ligase solution to each spot. Incubate 30 min at 37 &#176;C in a pre-warmed humidified chamber.</li>
	<li>Wash the slides 2 times for 5 min with ~50-60 mL 1x in situ wash buffer A in a Coplin jar at RT with gentle agitation.</li>
	<li>Prepare the Amplification-Polymerase mixture on ice by diluting the Amplification stock solution 1:5 in water. Then add 0.5 &#956;L of polymerase to 39.5 &#956;L of 1x Amplification solution for each spot. 
	NOTE: The amplification reagents are light sensitive and should be protected against direct exposure to light. The amplification reagent comes in four different colors (RED: excitation 594 nm, emission 624 nm; FarRED: excitation 644 nm, emission 669 nm; ORANGE: excitation 554 nm, emission 576 nm; GREEN: excitation 501 nm, emission 523 nm), make sure that the microscopy setup available has suitable excitation properties and filters to image these colors.</li>
	<li>Tap off 1x in situ wash buffer A and add 40 &#956;L of Amplification-Polymerase mixture per spot. Incubate for 100 min at 37 &#176;C in a pre-warmed humidified chamber.</li>
</ol>
5. Mounting and Imaging
<ol>
	<li>Prepare 0.01x in situ wash buffer B by adding 1 mL of 1x Wash Buffer B to 99 mL of water.</li>
	<li>Tap off the Amplification-Polymerase mixture and wash slides 2 times for 10 min with 1x in situ wash buffer B in a Coplin jar at RT with gentle agitation.</li>
	<li>Wash the slides 1 min in 0.01x wash buffer B.</li>
	<li>Tap off excess 0.01x wash buffer B and dry the slides around the spots with a lint-free tissue. Remove as much liquid from the spot as possible using a pipet without touching the spot, or by tilting the slide.</li>
	<li>Dilute DAPI-containing mounting medium 1:5 in mounting medium without DAPI to avoid overstaining of the nuclei.</li>
	<li>Add 25-30 &#956;L of 1:5 DAPI-containing mounting medium to a 24 mm x 24 mm coverslip and pick up the cover slip with the slide, apply slight pressure to ensure that there are no trapped air bubbles. Seal the coverslip with nail polish and wait at least 15 min before imaging.</li>
	<li>Analyze the slides using a (confocal) fluorescence microscope.
	<ol>
		<li>Count the number of punctate PLA signals per cell in at least 4 acquired image fields (20x objective, counting at least 20 cells per condition or antibody combination, based on a power calculation using an anticipated mean of 7 &#177; 5 signals in the treated cells, and 2 &#177; 2 signals in the untreated cells, with a probability of a type-I error of 0.05, and a power of 80%, as reported previously15). 
		NOTE: The best resolution is achieved by acquisition of a Z-stack and deconvolution using software packages (e.g., ImageJ, the Leica Acquisition Suite (LAS)). The signals should be readily discernible within the nuclear perimeter, as defined by the DAPI staining.</li>
		<li>Do not count signals that clearly appear outside the nuclei. Typically, NCS treatment or &#947;-irradiation results in approximately 5-10 phosho-p53/ATM PLA signals per cell. The &#39;single-antibody&#39; controls may give some background signal, but this should not exceed 1-2 PLA signals per 1 out of every 20 cells.</li>
		<li>For publication and presentation purposes, capture images using a 40X or 63X immersion oil objective. Slides can be stored at 4 &#176;C and should be protected from exposure to light.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this report, it is demonstrated that PLA can be used to determine and visualize the specific interaction between proteins in suspension cell cultures. Of note, the protocol described here is not restricted to the study of DNA repair complexes but also applies to visualize and quantify other protein-protein interactions in suspension cell cultures. It is shown that the ATM kinase interacts with phosphorylated p53 in G1-arrested BCR-ABL+ B-ALL cells when exposed to a DNA damage-inducing agent. Previously, the PLA techni...

DNA damage triggers a highly regulated series of events involving protein-protein interactions and post-translational modifications that ensures the efficient and rapid repair of DNA, thereby safeguarding genomic integrity1. Typically, DNA repair is studied in cells exposed to ionizing radiation by monitoring the formation of so-called ionizing radiation-induced foci (IRIF) by (confocal) fluorescence microscopy. Many DNA repair and DNA damage-sensing proteins form IRIFs, which represent protein complexes that nucleate at chromatin sites sustaining DNA damage2,3. The location and resolution of IRIFs over time gives important insight into spatiotemporal organization of DNA repair, and may indicate the involvement of different DNA repair pathways. The nature of the DNA damage and the cell-cycle stage in which the damage is attained determines which DNA repair pathway is activated. For instance, in cells actively engaged in DNA replication (S-phase), homologous recombination (HR) is the dominant DNA repair pathway, whereas in cells in the G1- or G2/M-phase of the cell-cycle, the non-homologous end-joining (NHEJ) repair pathway predominates. One of the earliest events following DNA damage is the activation of the DNA damage-sensing kinases Ataxia Telangiectasia-Mutated protein (ATM), which is mostly active in the G1- and G2/M-phases of the cell-cycle and regulates NHEJ, and Ataxia Telangiectasia and Rad3-related protein (ATR), which acts in S-phase by activating HR. Both ATM and ATR are very pleiotropic kinases that phosphorylate many proteins that are involved in DNA repair, cell death and survival4. Both kinases have been shown to phosphorylate and activate the tumor-suppressor protein p53 following the exposure to genotoxic stress, indicating that these kinases are upstream mediators of a pivotal tumor suppressive signaling axis5,6.
The formation and composition of IRIFs is typically assessed by determining co-localization of different proteins using dual-color immunofluorescence staining and microscopy, however, not all proteins that are part of repair protein complexes form IRIFs, which limits the applicability of this approach. Moreover, (confocal) immunofluorescence microscopy is limited by the diffraction properties of light, resulting in a rather poor spatial resolution of about 200-300 nm, exceeding the size of most subcellular structures, which essentially prohibits the direct interrogation of protein-protein interactions at the molecular level. As such, the co-localization of immunofluorescence staining patterns as detected by (confocal) fluorescence microscopy is not necessarily indicative for direct protein-protein interactions. Recently, new super-resolution technologies have been developed, such as three-dimensional structured illumination microscopy (3D-SIM)7, which was successfully used to study 53BP1 and BRCA1 IRIF formation at nano-scale detail, revealing the spatial distribution characteristics of these proteins that could not detected by confocal laser scanning microscopy8.
Several other methods can be used to detect protein-protein interactions in vivo, such as co-immunoprecipitation, pull-down methods and yeast two-hybrid screening approaches. However, these techniques are rather cumbersome, require large amounts of cells or proteins or involve overexpression of proteins, which introduces experimental artifacts. More recently, a novel technique has been developed that allows the visualization and quantification of protein-protein interactions in situ (i.e. in cells and in tissues), which is termed Proximity Ligation Assay (PLA)9,10. Primary antibodies that recognize two proteins of interest are detected by secondary antibodies that are conjugated to oligonucleotides (so-called PLA probes). If the two different secondary antibodies are sufficiently close due to interactions between the proteins recognized by the primary antibodies, the conjugated oligonucleotides hybridize and can be ligated to form a closed circular DNA substrate. This circular substrate is subsequently amplified by rolling circle amplification, and visualized with fluorochrome-conjugated complementary oligonucleotides. Using PLA, the subcellular localization of the protein-protein interaction is preserved as the fluorescently labeled rolling circle amplification-product remains attached to the PLA probes. The resolution of this assay is &#60;50 nm, based on the finding that the diameter of an antibody is approximately 7-10 nm11. Rolling circle amplification can only take place in case two pairs of antibodies (primary + secondary) physically interact within the perimeter that is defined by their size (10 + 10 + 10 + 10 = 40 nm). The signal amplification step increases the sensitivity of the PLA assay and enables the detection of interactions of scarcely expressed proteins. PLA generates punctate, foci-like signals patterns that can be quantified on a per cell basis, by which the intra- and inter-cellular variation in protein-protein interactions can be assessed.
The formation and composition of DNA repair complexes and IRIFs is mostly studied in adherent cell lines such as the human bone osteosarcoma epithelial cell line U2OS, the human embryonic kidney cell line HEK293 and the retinal pigment epithelial cell line RPE-1, which are fast-growing and easy to transfect. Suspension cell cultures such a lymphoid and myeloid cell lines are used less frequently, as these are less amenable to transfection and generally do not adhere to coverslips, thus requiring additional/alternative steps for imaging. The resolution of DNA damage is however very relevant in the context of lymphoid and myeloid malignancies, as the DNA damage response is frequently affected by genomic (driver) aberrations in these tumors, playing a pivotal role in the malignant transformation of normal lymphoid and myeloid (progenitor) cells12,13,14.
This protocol describes how PLA can be used to assess and quantify protein-protein interactions following the induction of DNA damage in suspension cell cultures. Here, PLA is performed to determine and visualize the interactions between ATM and p53 upon DNA damage in human B-cell leukemia cells that are induced to undergo a G1-phase cell-cycle arrest. Of note, the protocol presented here is not restricted to studying ATM and p53 interactions in G1-arrested leukemia cells, but can also be used to visualize other protein-protein interactions in various cell types and suspension cell cultures.

<table><tbody><tr><td>BV173 cell line</td><td>DSMZ</td><td>AC-20</td><td>BCR-ABL+ B-ALL cell line</td></tr><tr><td>SUP-B15 cell line</td><td>DSMZ</td><td>ACC-389</td><td>BCR-ABL+ B-ALL cell line</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Gibco (Life Technologies)</td><td>21980-032</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Sigma Aldrich</td><td>F7524</td><td>lot #: 064M3396</td></tr><tr><td>L-glutamine</td><td>Gibco (Life Technologies)</td><td>25030-024</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco (Life Technologies)</td><td>15140-122</td><td></td></tr><tr><td>imatinib methanesulfonate</td><td>LC Laboratories</td><td>I-5508</td><td>Dissolve in DMSO, prepare 10 mM stock solution</td></tr><tr><td>neocarzinostatin</td><td>Sigma Aldrich</td><td>N9162</td><td>Mutagenic/teratogenic, handle with care</td></tr><tr><td>KU55933</td><td>Selleckchem</td><td>S1092</td><td>Dissolve in DMSO, prepare 5 mM stock solution</td></tr><tr><td>Starfrost Microscopy Slides</td><td>Waldemar Knittel</td><td>VA11200 003FKB</td><td></td></tr><tr><td>PAP pen liquid blocker</td><td>Sigma Aldrich</td><td>Z377821-1EA</td><td></td></tr><tr><td>Cytospin funnel</td><td>Q Path Labonord SAS</td><td>003411324</td><td></td></tr><tr><td>Duolink In Situ Red Starter Kit Goat/Rabbit</td><td>Sigma Aldrich</td><td>DUO92105</td><td>Available for different species/combinations, also available in FarRED, Orange and Green</td></tr><tr><td>goat-anti-ATM</td><td>Bethyl Laboratories</td><td>A300-136A</td><td>PLA-grade; we succesfully used lot#A300-136A-1 in our studies</td></tr><tr><td>rabbit-anti-phospho-Ser15-p53</td><td>Cell Signaling Technology</td><td>9284</td><td>We succesfully used lot #9284-4 in our studies</td></tr><tr><td>Vectashield antifading mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Vectashield antifading mounting medium</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>4% paraformaldehyde in PBS</td><td>Santa Cruz Biotechnology</td><td>sc-281692</td><td>Also available from various other vendors</td></tr></tbody></table>

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.

Phosphorylation of p53 at residue Ser15 was shown to be dependent on ATM kinase activity16. To demonstrate and confirm the specificity of the PLA technique on cytospin preparations of suspension cell cultures, it is shown that induction of DNA damage by 2 h NCS treatment of BCR-ABL+ B-ALL cells arrested in the G1-phase of the cell-cycle resulted in the specific interaction between ATM and phospho-Ser15-p53, as expected. Punctate PLA signals were observed in the nuc...

Watch this Scientific Journal Video about Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay at JoVE.com

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

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 ...

detection-visualization-dna-damage-induced-protein-complexes

Research

JoVE Journal

Biochemistry

9.9K Views.  Academic Medical Center, University of Amsterdam, Lymphoma and Myeloma Center Amsterdam (LYMMCARE). 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. 

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

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Bioengineering

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

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

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

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

Genetics

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Neuroscience

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Cancer Research

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Biology

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Assessing Protein Interaction and Localization in DNA Damage

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

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

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

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

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

Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

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

Test ligacji zbliżeniowej do badania interakcji białko-białko in situ

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

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

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In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

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

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

Test ligacji zbliżeniowej do badania interakcji białko-białko in situ