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

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.