Martucci Martial, Debar Louis, van den Wildenberg Siet, Farge Geraldine
Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France.
Université Clermont Auvergne, CNRS, IRD, Université Jean Monnet Saint Etienne, LMV, Clermont-Ferrand, France.
Methods Mol Biol. 2023;2615:121-137. doi: 10.1007/978-1-0716-2922-2_10.
Mitochondrial transcription factor A (TFAM) plays a key role in the organization and compaction of the mitochondrial genome. However, there are only a few simple and accessible methods available to observe and quantify TFAM-dependent DNA compaction. Acoustic Force Spectroscopy (AFS) is a straightforward single-molecule force spectroscopy technique. It allows one to track many individual protein-DNA complexes in parallel and to quantify their mechanical properties. Total internal reflection fluorescence (TIRF) microscopy is a high-throughput single-molecule technique that permits the real-time visualization of the dynamics of TFAM on DNA, parameters inaccessible with classical biochemistry tools. Here we describe, in detail, how to set up, perform, and analyze AFS and TIRF measurements to study DNA compaction by TFAM.
线粒体转录因子A（TFAM）在线粒体基因组的组织和压缩过程中起着关键作用。然而，目前仅有少数简单且易于操作的方法可用于观察和量化TFAM依赖的DNA压缩。声镊光谱技术（AFS）是一种直接的单分子力谱技术。它能够使人们并行追踪多个单独的蛋白质-DNA复合物，并对其力学性质进行量化。全内反射荧光（TIRF）显微镜是一种高通量单分子技术，可实时可视化TFAM在DNA上的动态变化，而这些参数是传统生物化学工具无法获取的。在此，我们详细描述如何设置、进行和分析AFS及TIRF测量，以研究TFAM介导的DNA压缩。
