Vizjak Petra, Hepp Nicola, Mueller-Planitz Felix
Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.
Department of Molecular Biology, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.
Methods Mol Biol. 2025;2881:271-291. doi: 10.1007/978-1-0716-4280-1_14.
Adenosin triphosphate (ATP)-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit, evict, or reposition nucleosomes along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. Both assays exploit the changes in the accessibility of DNA to restriction enzymes during the remodeling reaction. The first assay uses the restriction enzyme to cleave the restriction site as soon as it becomes accessible during remodeling. As such, this assay mostly reports the kinetic parameter of the "forward" reaction of nucleosome remodeling. In contrast, the second assay measures how fast a particular nucleosome in the array reaches its steady-state position.
三磷酸腺苷（ATP）依赖的核小体重塑因子塑造了真核染色质的核小体景观。它们沿着DNA沉积、移除或重新定位核小体，这一过程称为核小体滑动。传统上，重塑是使用单核小体作为模型底物进行分析的。然而，在体内，核小体形成具有规则间距的延伸阵列。在这里，我们描述了如何在体外重建规则间距的核小体阵列，以及如何使用这些阵列在试管中剖析重塑过程。我们概述了两种测定方法。这两种测定方法都利用了重塑反应过程中DNA对限制酶可及性的变化。第一种测定方法是在重塑过程中限制位点一旦变得可及，就使用限制酶切割该限制位点。因此，这种测定方法主要报告核小体重塑“正向”反应的动力学参数。相比之下，第二种测定方法测量阵列中特定核小体达到其稳态位置的速度。
