We thank Funda&#231;&#227;o de Amparo a Pesquisa do Estado de S&#227;o Paulo (FAPESP, through Grant Tem&#225;tico 2022/15126-9 to JK and fellowship 21/09439-1 to LARM) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnol&#243;gico (CNPq) for funding this research.

1. Cell plating
NOTE: Cells can be seeded in microscopic fluorescence slides, chambers, or plates. The use of small coverslips or 96/ 384 well plates is recommended for testing multiple conditions and spare reagents. The use of coverslips is advisable for cell lines that detach easily, like HEK293 cells. The coverslips can be previously coated with a poly-L-lysine solution to improve the attachment.
<ol>
	<li>Plate cells considering a vehicle, technical controls, and biologi...

Assessing Protein Interaction and Localization in DNA Damage

<ol>
	<li>Xiao, W., Samson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+evidence+for+endogenous+DNA+alkylation+damage+as+a+source+of+spontaneous+mutation+in+eukaryotic+cells.">In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells.</a> Proc Nat Acad Sci U S A. 90 (6), 2117-2121 (1993).</li><li>Lindahl, T., Barnes, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+endogenous+DNA+damage.">Repair of endogenous DNA damage.</a> Cold Spring Harb Symp Quant Biol. 65, 127-134 (2000).</li><li>Hoeijmakers Jan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage,+aging,+and+cancer.">DNA damage, aging, and cancer.</a> N Engl J Med. 361 (15), 1475-1485 (2009).</li><li>Giglia-Mari, G., Zotter, A., Vermeulen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+response.">DNA damage response.</a> Cold Spring Harb Perspect Biol. 3 (1), a000745 (2011).</li><li>Ciccia, A., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+damage+response:+Making+it+safe+to+play+with+knives.">The DNA damage response: Making it safe to play with knives.</a> Mol Cell. 40 (2), 179-204 (2010).</li><li>Chatterjee, N., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+DNA+damage,+repair+and+mutagenesis.">Mechanisms of DNA damage, repair and mutagenesis.</a> EnvironMol Mutagen. 58 (5), 235-263 (2017).</li><li>Mah, L. J., El-Osta, A., Karagiannis, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GammaH2AX:+a+sensitive+molecular+marker+of+DNA+damage+and+repair.">GammaH2AX: a sensitive molecular marker of DNA damage and repair.</a> Leukemia. 24 (4), 679-686 (2010).</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> Curr Biol. 10 (15), 886-895 (2000).</li><li>Nakamura, A. J., Rao, V. A., Pommier, Y., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complexity+of+phosphorylated+H2AX+foci+formation+and+DNA+repair+assembly+at+DNA+double-strand+breaks.">The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks.</a> Cell cycle (Georgetown, Tex). 9 (2), 389-397 (2010).</li><li>Karchugina, S., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+heterodimerization+of+protein+isoforms+using+an+in+situ+proximity+ligation+assay.">Detection of heterodimerization of protein isoforms using an in situ proximity ligation assay.</a> J Vis Exp. (140), (2018).</li><li>Tubbs, E., Rieusset, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+endoplasmic+reticulum+and+mitochondria+interactions+by+in+situ+proximity+ligation+assay+in+fixed+cells.">Study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells.</a> J Vis Exp. (118), (2016).</li><li>Nguyen, C. L., 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=Nek4+regulates+entry+into+replicative+senescence+and+the+response+to+DNA+damage+in+human+fibroblasts.">Nek4 regulates entry into replicative senescence and the response to DNA damage in human fibroblasts.</a> Mol CellBiol. 32 (19), 3963-3977 (2012).</li><li>Basei, F. L., 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=New+interaction+partners+for+Nek4.1+and+Nek4.2+isoforms:+from+the+DNA+damage+response+to+RNA+splicing.">New interaction partners for Nek4.1 and Nek4.2 isoforms: from the DNA damage response to RNA splicing.</a> Proteome Sci. 13 (1), 11 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal Cell Culture Guide. , (2024).</li><li>Soubeyrand, S., Pope, L., Hach&#233;, R. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+II&#945;-dependent+induction+of+a+persistent+DNA+damage+response+in+response+to+transient+etoposide+exposure.">Topoisomerase II&#945;-dependent induction of a persistent DNA damage response in response to transient etoposide exposure.</a> MolOncol. 4 (1), 38-51 (2010).</li><li>Wei, F., 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=Etoposide-induced+DNA+damage+affects+multiple+cellular+pathways+in+addition+to+DNA+damage+response.">Etoposide-induced DNA damage affects multiple cellular pathways in addition to DNA damage response.</a> Oncotarget. 9 (35), 24122-24139 (2018).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Melo-Hanchuk, T. D., Slepicka, P. F., Pelegrini, A. L., Menck, C. F. M., Kobarg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NEK5+interacts+with+topoisomerase+II&#946;+and+is+involved+in+the+DNA+damage+response+induced+by+etoposide.">NEK5 interacts with topoisomerase II&#946; and is involved in the DNA damage response induced by etoposide.</a> J Cell Biochem. 120 (10), 16853-16866 (2019).</li><li>George, J., Mittal, S., Kadamberi, I. P., Pradeep, S., Chaluvally-Raghavan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+proximity+ligation+assay+(PLA)+for+detection+of+RNA-protein+complex+interactions+in+cell+lines.">Optimized proximity ligation assay (PLA) for detection of RNA-protein complex interactions in cell lines.</a> STAR Protoc. 3 (2), 101340 (2022).</li><li>Zhang, W., Xie, M., Shu, M. D., Steitz, J. A., DiMaio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proximity-dependent+assay+for+specific+RNA-protein+interactions+in+intact+cells.">A proximity-dependent assay for specific RNA-protein interactions in intact cells.</a> RNA. 22 (11), 1785-1792 (2016).</li><li>Hegazy, M., 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=Proximity+ligation+assay+for+detecting+protein-protein+interactions+and+protein+modifications+in+cells+and+tissues+in+situ.">Proximity ligation assay for detecting protein-protein interactions and protein modifications in cells and tissues in situ.</a> Curr Protoc Cell Biol. 89 (1), e115 (2020).</li></ol>

The data show that the use of PLA concomitantly to a DNA-damaged marker can provide the most information in a DNA damage response profiling, showing the spatial and temporal behavior of the interaction after the insult. PLA is a versatile method that has been used for dimerization identification, organelles contact determination, protein-nucleic acid interaction, and mainly protein-protein interaction10,11,19,<sup cla...

Cellular DNA damage occurs daily because of the spontaneous chemical reactions and is also increased by exogenous factors such as genotoxic agents (radiation and chemicals such as etoposide) and oxidative stress1,2,3. The cells have a complex machinery that corrects a myriad of different types of DNA damage, from removal of bases to replicative fork torsion or interruption to the most deleterious lesion: the DNA double-strand break4,5.
Several proteins that participate in the DDR have already been characterized, and excellent revisions explore the pathways of non-homologous end join (NHEJ) and homologous recombination (HR), the main pathways implied in double-strand breaks (DSB) repair5,6. The phosphorylation of the histone H2A variant, H2AX, at serine 139 (thereafter named y-H2AX) has been used for many years as a sensitive and reliable marker of the double-strand break. The phosphorylation of H2AX is observed in the first minutes following the damage and can persist for hours7.
The advantage of using the immunofluorescence detection of &#947;-H2AX foci is the characterization of the temporal and spatial distribution of the foci, as well as its co-staining with other proteins. Moreover, immunofluorescence does not require lysis, which preserves cellular context information and makes it more informative. Besides, as &#947;-H2AX is formed de novo, it is not abundantly present in the absence of DNA damage, making it a specific marker7.
H2AX is mostly phosphorylated by protein kinase ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), the sensors of DSBs. Ku70/80 (XRCC6 X-ray repair cross-complementing 6/ XRCC5 X-ray repair cross-complementing 5) and DNA-protein kinase catalytic subunit (DNA-PKcs) are responsible for DSB identification and the protection of DNA ends, whereas Artemis carries out end-processing to facilitate ligation by the XRCC4-Ligase IV complex. The phosphorylation of H2AX at sites flanking DSBs acts as a signal for the recruitment of several repair proteins and signaling for cell cycle arrest, death, or survival8.
The analysis of changes in y-H2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response. Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the co-localization of the protein of interest with y-H2AX foci. However, except for the new super-resolution fluorescence methods, concluding the local interaction with DNA damage sites can be tricky. Furthermore, immunofluorescence detection usually depends on many protein molecules at the DNA damage site, making it difficult to identify scarce proteins9.
Here, we show an assay to evaluate the localization of proteins involved in the DDR pathway using y-H2AX as a marker of the damage site. This assay can be used to characterize temporal localization under different insults that cause DNA damage.
The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures10,11and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is like conventional immunofluorescence and allows the simultaneous staining of organelle or cellular structure-specific markers, such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX. The use of PLA allows the identification of protein interaction during DNA damage in a sensitive, specific, and reliable manner that can be used to monitor the interaction during the cell cycle, in response to different stimuli, and at different times after genotoxic stress.
We show here the use of PLA concomitantly to conventional &#947;-H2AX immunofluorescence to localize Nek4-Ku70 interaction after etoposide-induced DNA damage. Nek4, a Nek (NIMA-related kinases) family member, has no clear role in DNA Damage response. In 2012, Nguyen and colleagues showed that Nek4 interacts with Ku70/Ku80 proteins, and in the Nek4 absence, these proteins are not recruited to the DNA damage site, and H2AX phosphorylation is impaired12. The cellular localization of this interaction is unknown so far. We have observed a reduction in Ku70 phosphorylation in cells expressing Nek4 kinase-dead mutant13but whether Nek4 phosphorylates directly Ku70 remains to be elucidated. Considering that Nek4 interaction with Ku70 is increased after etoposide treatment according to immunoprecipitation studies12, we attempt to localize this interaction using PLA and the &#947;-H2AX marker.
Usually, the interaction studies are based on immunoprecipitation assays, but these are in vitro and do not provide information about the location of the interaction. When possible, an immunoprecipitation of fractioned cells (nucleus, mitochondrial, cellular membrane, or cytosol) can be performed, but performing a time course of the interaction is costly and laborious. Immunofluorescence can give information about the cellular localization of the proteins, but proving the interaction can be difficult and depends on the resolution of the microscopes. Here, we show the advantage of using the Proximity Ligand Assay to monitor the interaction of two proteins in a DNA damage context.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Black 384-well plates</td>
			<td>Perkin Elmer Cell carrier plates</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey&#160; anti- Rabbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Duo link Donkey anti Mouse Minus</td>
			<td>Sigma</td>
			<td>DUO92004</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink antibody diluent</td>
			<td>Sigma</td>
			<td>DUO82008</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink blocking solution 1X</td>
			<td>Sigma</td>
			<td>DUO82007</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Detection reagent Far red</td>
			<td>Sigma</td>
			<td>DUO92013</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Donkey anti goat plus</td>
			<td>Sigma</td>
			<td>DUO92003</td>
			<td></td>
		</tr>
		<tr>
			<td>Duolink Donkey anti rabbit plus</td>
			<td>Sigma</td>
			<td>DUO92002</td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Sigma</td>
			<td>E1383</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Nek4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-5517</td>
			<td>goat anti Nek4 was used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo</td>
			<td>H1399</td>
			<td>0.6 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Leica DMI microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti Ku70</td>
			<td>Thermo</td>
			<td>MA5-13110</td>
			<td>mouse anti Ku70 was used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Mouse anti TOPII&#946;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-365071</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>Rabbit anti Nek5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-84527</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>Rabbit anti Y H2AX</td>
			<td>Cell Signalling</td>
			<td>9718S</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>U2OS cell line</td>
			<td>ATCC</td>
			<td>HTB-96&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a cellular system that maintains genome stability and integrity. Dysfunctions in this process cause several disease such as cancer, age-related disease, and chronic inflammation. Our goal is to identify proteins that cooperate in this process, which allows the identification of alternative targets for therapeutic integration.To characterize a protein's participation in the DNA damage response, immunofluorescence studies with the phosphorylated form of the H2AX histone, a marker of DNA damage, are usually applied. Moreover, protein collection assays such as co-immunoprecipitation can be performed after DNA damage to identify the protein's role in signaling. Interaction studies are usually based on immunoprecipitation assays, but these are in vitro and do not provide information about the location of the interaction.Immunofluorescence can give information about the cell localization of the proteins, but proving the interaction can be difficult and depends on the resolution of the microscope. We show here that a simple protocol combining proximity ligand assay with Gamma-H2AX staining allows the spatial and temporal characterizations of proteins'interaction in DNA damage context. Moreover, we have provided a user-friendly macro for data analysis.

We have observed Nek4-Ku70 interaction in the absence of etoposide treatment. However, this interaction can occur outside of the nucleus (Figure 1A). The Nek4-Ku70 interaction increases after DNA damage and is concentrated in the nucleus (Figure 1A). In the case of Nek5-Topoisomerase II &#946; (TOPII&#946;) interaction, used in this assay as a positive control based on literature results18, the interaction is greatly increased by etoposid...

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-ligand-assay-to-localize-proteins-in-dna-damage-sites

Research

JoVE Journal

Biology

189 vistas.  University of Campinas. La respuesta al daño del ADN es un sistema celular que mantiene la estabilidad e integridad del genoma. Las disfunciones en este proceso causan varias enfermedades, como el cáncer, las enfermedades relacionadas con la edad y la inflamación crónica. Nuestro objetivo es identificar proteínas que cooperen en este proceso, lo que permite la identificación de dianas alternativas para la integración terapéutica.Para caracterizar la participación de ...

Video: Autor destacado: Combinación del ensayo de ligando de proximidad con la tinción Gamma-H2AX para caracterizar las interacciones de proteínas en la respuesta al daño