DNA损伤响应和修复蛋白聚点机理研究

项目名称： DNA损伤响应和修复蛋白聚点机理研究

项目编号： No.31200630

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 生物物理、生化与生物分子学、生物力学与组织工程

项目作者： 杜广华

作者单位： 中国科学院近代物理研究所

项目金额： 23万元

中文摘要： 生命体DNA受到破坏发生双链断裂后，会引起DNA损伤和修复机制的一系列级联响应，许多相关蛋白被激活、修饰并在DNA损伤处形成蛋白聚点以开展修复进程。免疫荧光显微观测可见些蛋白聚点成微米大小的亮点。这些蛋白聚点的内部结构和蛋白分布对于研究DNA损伤和修复机理、相关蛋白角色和相互作用、肿瘤放射治疗和辐射防护具有重要意义。但是由于受传统光学显微镜的分辨率限制，无法获得高清晰的聚点图像，聚点的内部信息至今仍处于未知状态。本项目使用X射线和高能重离子辐照MRC5与HeLa细胞，用可控开关荧光分子染色γ-H2AX和53BP1蛋白，采用最新的超分辨显微技术STORM来获取这些蛋白聚点的单分子纳米超分辨图像，研究DNA损伤修复聚点的高清结构及γ-H2AX和53BP1蛋白在聚点内的分布，研究聚点形态和结构与DNA修复动态、细胞周期等的关系及不同辐射品质下DNA损伤修复的特点，揭示DNA损伤修复机理。

中文关键词： DNA损伤修复；g-H2AX；53BP1；XRCC1；活细胞成像

英文摘要： A process cascade involved in DNA damage response and repair is initiated upon DNA double strands break damages induced by external stimuli; and many kinds of protein are activated, modified and accumulated at the DNA damage site to form protein foci and to repair the damage. Those protein foci show micrometer bright spots under fluorescence microscope after immuno-fluorescence staining. Although the inner structure of the protein foci and the protein distribution is very important in the study of DNA repair mechanism, roles and interaction of DNA damage response and repair proteins, and cancer treatment and radiation protection, the inner information of the foci keeps still unknown due to the resolution limit of traditional optical microscopy. In this proposal, we irradiate MRC-5 and HeLa cells, immuno-stained with γ-H2AX and 53BP1 antibodies and photon-switchable fluorescence molecules. Using the super-resolution microscopy technique STORM (stochastic optical reconstruction microscope) and single molecule fluorescence imaging, the high resolution image of the γ-H2AX and 53BP1 protein foci at the DSB sites, the inner foci structure and the sub-distribution of the γ-H2AX and 53BP1 protein in the foci, with relationship to DNA repair kinetics, cell cycle and radiation quality, will be studied to discover the mech

英文关键词： DNA damage and repair；g-H2AX；53BP1；XRCC1；live cell imaging