Nature. 2016 Jul 28;535(7613):566-9. doi: 10.1038/nature18643. Epub 2016 Jul 20.
Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. The structurally similar XRCC4 and XLF proteins are proposed to assemble as highly dynamic filaments at (or near) DSBs. Here we show, using dual- and quadruple-trap optical tweezers combined with fluorescence microscopy, how human XRCC4, XLF and XRCC4-XLF complexes interact with DNA in real time. We find that XLF stimulates the binding of XRCC4 to DNA, forming heteromeric complexes that diffuse swiftly along the DNA. Moreover, we find that XRCC4-XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4-XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. Understanding the dynamics and regulation of this mechanism will lead to clarification of how NHEJ proteins are involved in generating chromosomal translocations.
非同源末端连接（NHEJ）是修复哺乳动物细胞中 DNA 双链断裂（DSB）的主要途径。例如，在适应性免疫系统中的基因片段重排过程中，或者通过癌症治疗剂，会形成这种断裂。尽管 NHEJ 机制的核心组件是已知的，但评估这些组件的特定作用以及将断裂的 DNA 片段聚集在一起的动力学仍然很困难。结构相似的 XRCC4 和 XLF 蛋白被提议在（或接近）DSB 处组装成高度动态的纤维。在这里，我们使用双和四陷阱光镊结合荧光显微镜，实时展示了人 XRCC4、XLF 和 XRCC4-XLF 复合物与 DNA 的相互作用。我们发现 XLF 刺激 XRCC4 与 DNA 的结合，形成迅速扩散沿 DNA 的异源二聚体复合物。此外，我们发现 XRCC4-XLF 复合物能够强有力地桥接两个独立的 DNA 分子，并且这些桥能够沿 DNA 滑动。这些观察结果表明，XRCC4-XLF 复合物在 DNA 周围形成可快速重新连接断裂末端并将它们保持在一起的移动套管状结构。了解这种机制的动力学和调节将有助于阐明 NHEJ 蛋白如何参与产生染色体易位。
