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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
Published on: June 9, 2017
Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity
Mahnoush Bahjat1, Timon A Bloedjes1, Amélie van der Veen1
1Department of Pathology, Academic Medical Center, University of Amsterdam, Lymphoma and Myeloma Center Amsterdam (LYMMCARE).
Abstract:
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.
Insights
The DNA damage response involves key kinases like ATM and ATR. In situ Proximity Ligation Assay (PLA) visualizes direct protein interactions, such as ATM and p53, within DNA repair pathways.
Area of Science:
- Cellular biology
- Molecular genetics
- Biochemistry
Background:
- The DNA damage response (DDR) is crucial for repairing DNA lesions from various sources.
- Key kinases, including Ataxia-Telangiectasia Mutated (ATM), ATM- and Rad3-Related (ATR), and DNA-dependent Protein Kinase (DNA-PKcs), regulate DDR pathways.
- ATM and ATR activate p53, a tumor suppressor, through phosphorylation, impacting apoptosis and cell survival.
Purpose of the Study:
- To introduce and demonstrate the utility of in situ Proximity Ligation Assay (PLA) for studying protein-protein interactions in the DDR.
- To visualize direct interactions between proteins involved in DNA repair pathways with high specificity and sensitivity.
Main Methods:
- Utilized in situ Proximity Ligation Assay (PLA) on suspension cell cultures.
- Employed antibodies specific to proteins of interest, triggering an amplification reaction when proteins are within ~40 nm.
- Visualized protein-protein interactions using fluorescent labeling.
Main Results:
- Demonstrated the successful application of PLA to visualize direct protein-protein interactions in the DDR.
- Showcased the functional interaction between ATM and p53 as a model system.
- Highlighted PLA's ability to overcome the resolution limitations of traditional fluorescence microscopy for studying protein complexes.
Conclusions:
- In situ PLA is a powerful technique for directly visualizing protein-protein interactions in cellular and tissue contexts.
- PLA offers enhanced specificity and sensitivity for studying DDR components, such as the ATM-p53 interaction.
- This method provides valuable insights into the molecular mechanisms underlying DNA repair and cellular responses to damage.
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