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.