ssDNA-PLA, a proximity ligation assay to interrogate DNA damage response proteins involved in homologous recombination

DNA end resection is critical for DNA double-strand break repair via homologous recombination (HR) and replication-coupled repair. Traditional approaches for detecting DNA end resection in cells include fluorescence imaging for RPA foci, BrdU labeling followed by anti-BrdU staining under native conditions to detect ssDNA, and quantitative PCR to detect ssDNA using the ER-AsiSI U2OS cell system. Here, we comprehensively examined a proximity ligation assay (PLA)- based approach, named ssDNA-PLA, to detect protein-ssDNA interaction by combining BrdU genome-wide DNA labeling with PLA using anti-BrdU and antibody against proteins of interest. We showed that the ssDNA-PLA method is a robust and reliable approach to detect proteins interacting with ssDNA in cells in response to DNA damage induced by various agents and replication stress, including known ssDNA binding proteins RPA, RAD51 and BLM. This approach can be used for studying proteins proximity to ssDNA that play roles in DNA end resection and HR repair.