Study of the Dynamic Changes of Double-Strand Break Sensor Proteins Interactome during DNA Damage Response

DNA double-strand breaks (DSB) are critical genomic lesions, and reduced DSB repair efficiency is associated with age-related disease development, like Alzheimer’s and Parkinson´s. Once DSB occurs, the DNA Damage Response (DDR) is initiated by binding of DSB sensor to the damaged DNA. During this response, there is massive recruitment of signaling proteins, chromatin remodelers, and effector molecules that will cope with the lesion. Cells have two main DSB-repair pathways, an error-free repair known as homology recombination (HR) and non-homologous end joining (NHEJ), which usually generates mutations. Both mechanisms are selectively used, and the activity of the sensor proteins arriving at the DSB site has a major role in the repair outcome. We investigated the sensor-dependent interaction networks of three sensors in a time-dependent manner (Sirt6, Ku80, and NBS1). Using a proximity-labeling assay (BioID) and IP-mass spec, we identified chromatin networks for each sensor during different DDR time points. Our results showed that after DSB induction, the basal interactome of each sensor protein changes, progressively incorporating new proteins and approaching a similar state that requires functions like Nucleolar metabolism, RNA processing and degradation, and chromatin remodeling. Each sensor has its independent interactome and the shared interactome of DDR repair proteins. Interestingly, after DSB induction Sirt6 recruits several epigenetic regulators absent in the Ku80 interactome that allows it to regulate the sites of damage epigenetically.