Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions
Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions
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DOI：
10.3791/55999
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发表时间：
2017-11-01
影响因子：
1.2
通讯作者：
Bartova, Eva
中科院分区：
文献类型：
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作者：
Legartova, Sona;Suchankova, Jana;Bartova, Eva
激光局部微照射是研究活细胞 DNA 修复相关过程的有用工具。在这里，我们描述了一种分析 DNA 损伤随时间变化的蛋白质动力学或局部微照射染色质上蛋白质-蛋白质相互作用的方法。我们还展示了如何使用 Fucci 细胞系统识别细胞周期的各个阶段，以研究 DNA 损伤处的细胞周期依赖性蛋白质动力学。还介绍了使用两种紫外激光（355 nm 和 405 nm）诱导不同类型 DNA 损伤的方法学描述。只有经 405 nm 二极管激光微照射的细胞才能正常进行有丝分裂，并且不含环丁烷嘧啶二聚体 (CPD)。我们还展示了如何在给定时间点固定微照射细胞以对感兴趣的内源蛋白进行免疫检测。对于 DNA 修复研究，我们还描述了在具有自发性 DNA 损伤灶的细胞中使用生物物理方法，包括 FRAP（光漂白后荧光恢复）和 FLIM（荧光寿命成像显微镜）。我们还展示了 FLIM-FRET（荧光共振能量转移）在蛋白质-蛋白质相互作用实验研究中的应用。
Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.