Klocko Andrew D, Crafton Kaleena M, Walsh Brian W, Lenhart Justin S, Simmons Lyle A
Department of Molecular, Cellular, and Developmental Biology, University of Michigan-Ann Arbor, USA.
J Vis Exp. 2010 Feb 8(36):1736. doi: 10.3791/1736.
Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.
原核生物和真核生物都会通过一系列复杂的生理变化来应对DNA损伤。多种DNA损伤和错配的DNA碱基对可刺激基因表达的改变、现有蛋白质的重新分布以及新蛋白质复合物的组装。荧光显微镜已成为一种强大的实验工具，用于可视化和量化这些以及对DNA损伤的其他反应，并监测活细胞复杂亚细胞结构内的DNA复制状态。荧光报告蛋白与DNA复制和修复机制的组分之间的翻译融合已被用于确定在体内将DNA修复蛋白靶向其同源损伤的线索，并了解这些蛋白在细菌细胞内是如何组织的。此外，与DNA损伤诱导型启动子相连的转录和翻译融合揭示了群体中的哪些细胞已激活遗传毒性应激反应。在本综述中，我们提供了一份详细的方案，用于使用荧光显微镜对单个细菌细胞中的DNA修复和DNA复制复合物的组装进行成像。特别是，这项工作聚焦于对枯草芽孢杆菌中的错配修复蛋白、同源重组、DNA复制和一种SOS诱导蛋白进行成像。这里描述的所有程序都很容易适用于对多种细菌物种中的蛋白质复合物进行成像。
