Abramson Family Cancer Research Institute and Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Pathology and Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA.
Mol Cell. 2018 Oct 18;72(2):222-238.e11. doi: 10.1016/j.molcel.2018.08.047. Epub 2018 Oct 4.
DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.
DNA 聚合酶停滞会激活 ATR 检查点激酶，进而抑制叉崩溃和断裂。在此，我们描述了使用 ATR 抑制 (ATRi) 作为一种识别小鼠和人类细胞中存在问题的 DNA 复制的基因组位点的方法。使用两种不同的方法确定了超过 500 个高分辨率的 ATR 依赖性位点：复制蛋白 A (RPA)-染色质免疫沉淀 (ChIP) 和 TdT 标记识别的断裂 (BrITL)。与 ATR 依赖性最相关的基因组特征是具有高结构形成潜力的重复 DNA。最依赖 ATR 稳定的重复包括结构形成的微卫星、反转录元件重复序列和准回文富含 AT 的重复序列。值得注意的是，这些不同类别的重复序列在它们形成的结构以及它们刺激 RPA 积累和断裂的能力方面存在差异，这表明在 ATR 抑制下复制叉崩溃的原因和特征可能以 DNA 结构特异性的方式发生变化。总之，这些研究确定了依赖 ATR 稳定的关键内源性复制应激源。
