Amitani Ichiro, Liu Bian, Dombrowski Christopher C, Baskin Ronald J, Kowalczykowski Stephen C
Department of Microbiology, University of California, Davis, California, USA.
Methods Enzymol. 2010;472:261-91. doi: 10.1016/S0076-6879(10)72007-3.
In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity.
在传统的生化实验中，单个蛋白质的行为会被总体平均所掩盖。为了更好地理解与DNA结合和/或在DNA上转运的蛋白质的行为，我们开发了一种仪器，该仪器利用光镊、微流控溶液输送和荧光显微镜来可视化单个蛋白质或作用于单个DNA分子的蛋白质组装体。一般的实验设计包括将单个DNA分子连接到聚苯乙烯微球上，然后将其用作微观手柄，用激光阱操纵单个DNA分子。通过对感兴趣的DNA或蛋白质进行荧光标记，然后使用高灵敏度荧光显微镜进行直接成像来实现可视化。我们描述了用于可视化单个蛋白质与单个DNA分子相互作用的样品制备和仪器。作为例子，我们描述了这些方法在参与重组介导的DNA修复的蛋白质研究中的应用，重组介导的DNA修复是维持基因组完整性所必需的过程。
