XRCC4-Ligase 4连接酶复合物与双链DNA动态交互作用的单分子荧光分析

XRCC4-Ligase 4（X4L4）是DNA双链断裂非同源末端连接（NHEJ）修复通路特有的连接酶复合物，其功能缺失会导致免疫缺陷性疾病。X4L4参与NHEJ双链DNA（dsDNA）联会及末端共价连接过程，但尚不清楚X4L4是如何与dsDNA动态相互作用进而形成何种形式的复合物参与这些过程。本项目拟发展生物化学单分子荧光方法，在单分子水平上研究X4L4与dsDNA的动态结合，以深入理解X4L4介导NHEJ的分子机制。表达纯化高荧光标记效率及高活性的X4L4蛋白，发展单分子两色荧光方法包括荧光共定位、荧光共振能量转移及荧光分步漂白等方法研究荧光标记的X4L4在单一dsDNA上的动态结合及形成复合物的结构，探讨NHEJ因子对X4L4-dsDNA动态交互作用的影响。在此基础上，进一步发展单分子三色荧光共定位方法研究介导两条dsDNA联会及末端共价连接的X4L4-dsDNA复合物形式。

XRCC4-Ligase 4 (X4L4) is the sole ligase complex specifically functioning in the non-homologous end joining (NHEJ) pathway for DNA double-stranded break (DSB) repair. X4L4 with a functional deficiency can lead to immunodeficiency. X4L4 is mainly involved in NHEJ dsDNA synapsis and covalent end-ligation. Currently, the active form of the X4L4-dsDNA complex formed from dynamic interactions between X4L4 and dsDNA in synapsis and end ligation remains unclear. This project will focus on the development of varied biochemical single-molecule fluorescence assays, which will be applied to study the dynamic binding of X4L4 to the dsDNA and further explore the molecular roles of X4L4 in the NHEJ process. Highly active and fluorescently labeled X4L4 will be first prepared. Two-color single-molecule fluorescence assays including fluorescence colocalization (CL), Förster resonance energy transfer (FRET), and stepwise fluorescence bleaching, will then be developed by using the fluorescent X4L4 and dsDNA to study the dynamic binding of X4L4 to a single dsDNA, the structures of formed X4L4-dsDNA complexes, and the effects of other NHEJ factors on the interactions of X4L4 and dsDNA. Based on the results above, a three-color single-molecule CL assay will be further developed to explore the active form of X4L4-dsDNA complex functioning in dsDNA synapsis and ligation.