Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX.
Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX
J Cell Biol. 2018 Apr 2;217(4):1521-1536. doi: 10.1083/jcb.201709121. Epub 2018 Feb 23.
DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding. Here we describe a sensitive single-cell-level assay system for the quantitative analysis of protein interactions with nascent DNA. Specifically, we achieve robust in situ analysis of protein interactions at DNA replication forks (SIRF) using proximity ligation coupled with 5'-ethylene-2'-deoxyuridine click chemistry suitable for multiparameter analysis in heterogeneous cell populations. We provide validation data for sensitivity, accuracy, proximity, and quantitation. Using SIRF, we obtained new insight on the regulation of pathway choice by 53BP1 at transiently stalled replication forks.
DNA 复制反应是多种细胞过程的核心，包括发育、癌症病因、药物治疗和耐药性。许多蛋白质和途径的存在是为了确保 DNA 复制保真度和保护停滞或受损的复制叉。一致地，参与 DNA 复制的蛋白质中的突变与多种疾病有关，包括胚胎发育和免疫缺陷、加速衰老、炎症增加、血液疾病和癌症。因此，用于在活跃和停滞的复制叉处有效定量分析蛋白质相互作用的工具对于先进和准确的生物学理解是关键。在这里，我们描述了一种用于定量分析与新生 DNA 相互作用的蛋白质的灵敏单细胞水平分析系统。具体来说，我们使用邻近连接结合 5'-乙烯-2'-脱氧尿苷点击化学在原位实现了对 DNA 复制叉处蛋白质相互作用（SIRF）的稳健分析，适用于异质细胞群体中的多参数分析。我们提供了关于灵敏度、准确性、接近性和定量的验证数据。使用 SIRF，我们获得了关于 53BP1 在瞬时停滞的复制叉处对途径选择的调控的新见解。
