Protocol for identifying DNA damage responders through proximity biotinylation of proliferating cell nuclear antigen interactors

TL;DR

This paper provides a detailed protocol to identify DNA repair proteins using TurboID proximity labeling and mass spectrometry.

Contribution

A new protocol for fusing PCNA with TurboID and using proximity labeling to identify DNA repair interactors.

Findings

The protocol enables efficient cell synchronization and DNA damage induction for proximity labeling.

Streptavidin pull-down combined with mass spectrometry identifies biotinylated PCNA interactors.

Abstract

TurboID-based proximity labeling is a powerful approach to capture protein-protein interactions within their native cellular environment. Here, we present a step-by-step protocol for fusing proliferating cell nuclear antigen (PCNA) to TurboID and generating stable cell lines via lentiviral transduction. We describe steps for cell synchronization, DNA damage induction, and proximity labeling, followed by fractionation, affinity purification, and mass spectrometry to identify biotinylated proteins.

For complete details on the use and execution of this protocol, please refer to Rona et al.1

•A TurboID-based proximity labeling protocol to identify proteins involved in DNA repair•Instructions for biochemical fractionation to reduce background and sample complexity•Steps to efficiently synchronize cells into different cell cycle phases•Streptavidin pull-down isolates biotinylated interactors for MS

A TurboID-based proximity labeling protocol to identify proteins involved in DNA repair

Instructions for biochemical fractionation to reduce background and sample complexity

Steps to efficiently synchronize cells into different cell cycle phases

Streptavidin pull-down isolates biotinylated interactors for MS

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

TurboID-based proximity labeling is a powerful approach to capture protein-protein interactions within their native cellular environment. Here, we present a step-by-step protocol for fusing proliferating cell nuclear antigen (PCNA) to TurboID and generating stable cell lines via lentiviral transduction. We describe steps for cell synchronization, DNA damage induction, and proximity labeling, followed by fractionation, affinity purification, and mass spectrometry to identify biotinylated proteins.

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