A Distinct Basal-Level Activity of ATR Facilitates Replication Fork Surveillance

Mammalian cells employ diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remains undefined. Here, we utilized Single-Molecule Localization Microscopy coupled with High-Order-Correlation image data-mining algorithms, to quantify the spatial configurations of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a distinct basal-level activity of ATR that monitors and regulates the amounts of RPA at forks to prevent its accumulation. Replication-stress amplifies the basal activity of ATR through increased volume of ATR-RPA interaction and enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication-stress response.