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 replication protein A foci, 5-bromo-2'-deoxyuridine (BrdU) labeling followed by anti-BrdU staining under native conditions to detect ssDNA, and quantitative PCR to detect single-stranded DNA (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 replication protein A, RAD51, and BLM. This approach can be used for studying the proximity of proteins to ssDNA that play roles in DNA end resection and HR repair. Keywords DNA damage repair, DNA end resection, PLA, ssDNA, RPA.