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

## TL;DR

The paper introduces ssDNA-PLA, a new method to study proteins that interact with single-stranded DNA during DNA repair.

## Contribution

ssDNA-PLA is a novel proximity ligation assay for detecting protein-ssDNA interactions in DNA repair.

## Key findings

- ssDNA-PLA reliably detects proteins interacting with ssDNA in response to DNA damage and replication stress.
- Known ssDNA-binding proteins like RPA, RAD51, and BLM were successfully identified using this method.
- The method is robust and applicable for studying DNA end resection and homologous recombination repair.

## Abstract

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