Triplex DNA structures, formed when a third DNA strand wraps around the major groove of DNA, are key molecular regulators and genomic threats. However, the regulatory network governing triplex DNA dynamics remains poorly understood. Here we reveal the binding and functional repertoire of proteins that interact with triplex DNA through chemoproteomic profiling in living cells. We develop a chemical probe that exhibits exceptional specificity towards triplex DNA. By employing a co-binding-mediated proximity capture strategy, we enrich triplex DNA interactome for quantitative proteomics analysis. This enables the identification of a comprehensive list of proteins that interact with triplex DNA, characterized by diverse binding properties and regulatory mechanisms in their native chromatin context. As a demonstration, we validate DDX3X as an ATP-independent triplex DNA helicase to unwind substrates with a 5′ overhang to prevent DNA damage. Overall, our study provides a valuable resource for exploring the biology and translational potential of triplex DNA.

当第三条 DNA 链缠绕在 DNA 的主要沟中时形成的三重 DNA 结构是关键的分子调节因子和基因组威胁。然而，控制三链体 DNA 动力学的调控网络仍然知之甚少。在这里，我们揭示了通过活细胞中的化学组学分析与三链 DNA 相互作用的蛋白质的结合和功能库。我们开发了一种化学探针，它对三链体 DNA 表现出非凡的特异性。通过采用共结合介导的邻近捕获策略，我们富集了三链体 DNA 相互作用组，用于定量蛋白质组学分析。这使得能够识别与三链体 DNA 相互作用的蛋白质的完整列表，这些蛋白质在其天然染色质环境中具有不同的结合特性和调节机制。作为演示，我们验证了 DDX3X 作为 ATP 非依赖性三链 DNA 解旋酶，以解开具有 5' 突出端的底物，以防止 DNA 损伤。总的来说，我们的研究为探索三链体 DNA 的生物学和转化潜力提供了宝贵的资源。