protocol for the detection of protein interaction at dna damage sites using proximity ligand assay

Assessing Protein Interaction and Localization in DNA Damage

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.

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.

Protocol for the Detection of Protein Interaction at DNA Damage Sites Using Proximity Ligand Assay

To begin, take cultured U2OS cells with 60%to 70%confluency and split them using 0.25%Trypsin-EDTA. Prepare 60 millimeter plates by adding round 13 millimeter cover slips. Plate 400, 000 cells in a 60 millimeter plate containing multiple 13 millimeter round cover slips.Then, remove the culture medium from the plates. Add culture medium containing 25 micromolar etoposide, and incubate cells for 20 minutes, 1 hour and 3 hours. After incubation, remove the medium from the plates.Wash the cells with PBS three times. To fix the cells, add ice cold methanol in enough volume to cover the cover slip surface, and incubate for 10 minutes at 20 degrees Celsius. Then, wash the cover slips with PBS three times and transfer them to a humidity chamber.After tapping off PBS, add 30 to 40 microliters of permeabilization buffer and incubate for 20 minutes at room temperature. After that, wash the cover slips three times with PBS. Then, tap off the PBS from the samples.Add 30 to 40 microliters of blocking solution provided in the PLA kit to each cover slip. Incubate the plate in a preheated humidity chamber at 37 degrees Celsius for 1 hour. Now dilute the primary antibodies in the antibody diluent provided in the kit.After tapping off the blocking solution, add the primary antibody solution to each cover slip and incubate at four degrees Celsius overnight in a humidity chamber. Dilute the minus and plus probes one to five in the antibody diluent. Use combinations like Donkey anti-Mouse MINUS with Donkey anti-Goat PLUS and Donkey anti-Mouse MINUS with Donkey anti-Rabbit PLUS.Now, tap off the primary antibody solution and wash once with TBST. After tapping off the excess TBST, add 20 microliters of the PLA probe solution to the cover slips. Then, incubate in the preheated humidity chamber at 37 degrees Celsius for 1 hour.Dilute the 5x ligation buffer in high purity water. Tap off the PLA probe solution and wash once with TBST. Now, add ligase to the ligation solution at 1:40 dilution immediately before applying it to the samples.Incubate in the preheated humidity chamber at 37 degrees Celsius for 30 minutes. Next, dilute the 5x amplification buffer in high purity water. Once the ligation solution is removed from the samples, wash the samples once with TBST.Add the polymerase to the amplification solution at 1:80 dilutions immediately before applying it to the samples. Incubate in the preheated humidity chamber at 37 degrees Celsius for 100 minutes. Tap off the amplification buffer, then wash two times with SSC buffer for 10 minutes each.After washing the samples two times with PBS, add the primary antibody solution to each cover slip and incubate at room temperature for 1 hour. At the end of the incubation, tap off the primary antibody solution and wash three times with TBST. Then, add the secondary antibody solution to each cover slip and incubate at room temperature for 20 minutes.Afterward, wash five times with TBST, two times with SSC buffer, and two times with 0.01x SSC buffer. Finally, acquire images from different wells or cover slips regions to avoid artifacts due to non homogeneous incubation, and save all the channels with the same name pattern.

protocol-for-detection-protein-interaction-at-dna-damage-sites-using

Research

JoVE Journal

Biology

31 visualizações. Para começar, pegue células U2OS cultivadas com 60% a 70% de confluência e divida-as usando 0,25% de tripsina-EDTA. Prepare placas de 60 milímetros adicionando lamínulas redondas de 13 milímetros. Placa 400.000 células em uma placa de 60 milímetros contendo várias lamínulas redondas de 13 milímetros.Em seguida, remova o meio de cultura das placas. Adicionar meio de cultura contendo 25 etoposídeo micromolar e incubar as células durante 20 m...