Nuclear reorganization, including the localization of proteins into discrete subnuclear foci, is a hallmark of the cellular response to DNA damage and replication stress. These foci are thought to represent transient environments or repair factories, in which the lesion is sequestered with molecules and co-factors that catalyze repair. For example, nuclear foci contain signaling proteins that recruit transducer proteins. One important class of transducers is the structure-selective endonucleases, such as SLX1-SLX4, MUS81-EME1, and XPF-ERCC1, which remove branched DNA structures that form during repair. The relocalization of structure-selective endonucleases into subnuclear foci provides a visual read-out for the presence of direct DNA damage, replication barriers, or DNA entanglements and can be monitored using fluorescence microscopy. By simultaneously probing for two or more fluorescent signals, fluorescence microscopy can also provide insights into the proximal association of proteins within a local environment. Here, we report an open-source and semi-automated method to detect and quantify subnuclear foci, as well as foci colocalization and the accompanying pixel-based colocalization metrics. We use this pipeline to show that pre-mitotic nuclei contain a basal threshold of foci marked by SLX1-SLX4, MUS81, or XPF. Some of these foci colocalize with FANCD2 and have a high degree of correlation and co-occurrence. We also show that pre-mitotic cells experiencing replication stress contain elevated levels of foci containing SLX1-SLX4 or XPF, but not MUS81. These results point towards a role for SLX1-SLX4 and XPF-ERCC1 in the early cellular response to replication stress. Nevertheless, most of the foci that form in response to replication stress contain either FANCD2 or one of the three endonucleases. Altogether, our work highlights the compositional heterogeneity of subnuclear foci that form in response to replication stress. We also describe a user-friendly pipeline that can be used to characterize these dynamic structures.

核重组，包括将蛋白质定位到离散的亚核病灶，是细胞对 DNA 损伤和复制应激反应的标志。这些病灶被认为代表了短暂的环境或修复工厂，其中病变与催化修复的分子和辅助因子隔离。例如，核病灶包含募集转导蛋白的信号蛋白。一类重要的传感器是结构选择性核酸内切酶，如 SLX1-SLX4、MUS81-EME1 和 XPF-ERCC1，它们去除修复过程中形成的分支 DNA 结构。结构选择性核酸内切酶重新定位到亚核病灶中，可以直观地读出直接 DNA 损伤、复制障碍或 DNA 缠结的存在，并可使用荧光显微镜进行监测。通过同时探测两个或多个荧光信号，荧光显微镜还可以深入了解局部环境中蛋白质的近端关联。在这里，我们报告了一种开源和半自动化的方法来检测和量化亚核焦点，以及焦点共定位和伴随的基于像素的共定位指标。我们使用此管道来显示有丝分裂前的细胞核包含由 SLX1-SLX4、MUS81 或 XPF 标记的病灶的基础阈值。其中一些病灶与 FANCD2 共定位，并且具有高度的相关性和共现性。我们还表明，经历复制应激的有丝分裂前细胞含有水平升高的含有 SLX1-SLX4 或 XPF 的病灶，但不含 MUS81。这些结果表明 SLX1-SLX4 和 XPF-ERCC1 在对复制应激的早期细胞反应中的作用。然而，大多数响应复制压力而形成的病灶包含 FANCD2 或三种核酸内切酶之一。总而言之，我们的工作突出了响应复制压力而形成的亚核病灶的组成异质性。我们还描述了一个用户友好的管道，可用于表征这些动态结构。