School of Life Science and Technology, Tokyo Institute of Technology, Oookayama, Meguro, Tokyo 152-8550, Japan.
Department of Chemical and Biological Engineering, Chemistry, Chalmers University of Technology, 412 96 Gothenburg, Sweden.
Int J Mol Sci. 2023 Nov 8;24(22):16092. doi: 10.3390/ijms242216092.
Linear dichroism (LD) is a differential polarized light absorption spectroscopy used for studying filamentous molecules such as DNA and protein filaments. In this study, we review the applications of LD for the analysis of DNA-protein interactions. LD signals can be measured in a solution by aligning the sample using flow-induced shear force or a strong electric field. The signal generated is related to the local orientation of chromophores, such as DNA bases, relative to the filament axis. LD can thus assess the tilt and roll of DNA bases and distinguish intercalating from groove-binding ligands. The intensity of the LD signal depends upon the degree of macroscopic orientation. Therefore, DNA shortening and bending can be detected by a decrease in LD signal intensity. As examples of LD applications, we present a kinetic study of DNA digestion by restriction enzymes and structural analyses of homologous recombination intermediates, i.e., RecA and Rad51 recombinase complexes with single-stranded DNA. LD shows that the DNA bases in these complexes are preferentially oriented perpendicular to the filament axis only in the presence of activators, suggesting the importance of organized base orientation for the reaction. LD measurements detect DNA bending by the CRP transcription activator protein, as well as by the UvrB DNA repair protein. LD can thus provide information about the structures of protein-DNA complexes under various conditions and in real time.
线性二色性（LD）是一种用于研究丝状分子（如 DNA 和蛋白质丝）的差分偏振光吸收光谱学。在本研究中，我们综述了 LD 在分析 DNA-蛋白质相互作用中的应用。通过使用流致剪切力或强电场对样品进行对齐，可以在溶液中测量 LD 信号。产生的信号与发色团（如 DNA 碱基）相对于丝轴的局部取向有关。因此，LD 可以评估 DNA 碱基的倾斜和滚动，并区分嵌入和沟槽结合配体。LD 信号的强度取决于宏观取向的程度。因此，通过 LD 信号强度的降低可以检测 DNA 的缩短和弯曲。作为 LD 应用的示例，我们介绍了限制酶切割 DNA 的动力学研究以及同源重组中间体（即 RecA 和 Rad51 重组酶与单链 DNA 的复合物）的结构分析。LD 表明，只有在激活剂存在的情况下，这些复合物中的 DNA 碱基才优先垂直于丝轴取向，这表明有序的碱基取向对反应很重要。LD 测量还检测到 CRP 转录激活蛋白和 UvrB DNA 修复蛋白引起的 DNA 弯曲。因此，LD 可以提供有关各种条件下和实时的蛋白质-DNA 复合物结构的信息。
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