Mammalian cells use 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, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven 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.

哺乳动物细胞使用多种途径来防止 DNA 复制过程中的有害后果，但细胞调查个体复制体以检测基础水平的自发复制障碍及其在复制压力期间的积累的机制仍未确定。在这里，我们使用单分子定位显微镜和高阶相关图像挖掘算法来量化在不受干扰的复制过程中和复制压力下单个细胞中单个复制体的组成。我们确定了 ATR 的基础水平活动，该活动在正常复制期间监控和调节叉处的 RPA 量。复制应力通过增加 ATR-RPA 相互作用的体积和扩散驱动的 ATR 在叉处的富集来放大基础活动。ATR 的这种局部拥挤提高了它的碰撞概率，刺激了它的复制应激反应的激活。最后，我们提供了一个计算模型，描述了 ATR 的基础活性如何被放大以产生其典型的复制应激反应。