Vitolo J M, Thiriet C, Hayes J J
University of Rochester Medical Center, Rochester, New York, USA.
Curr Protoc Mol Biol. 2001 May;Chapter 21:Unit 21.4. doi: 10.1002/0471142727.mb2104s48.
The native chromatin complex within most eukaryotic nuclei is very difficult to study by biochemical means, so researchers have developed methods for studying smaller portions of the complex. This unit details the use of DNase I and hydroxyl radicals to characterize histone-DNA interactions within such portions of the complex. DNase I digestion can be used to determine what regions of a DNA segment are intimately associated with the core histone proteins and what regions are more like naked DNA (i.e., linker DNA within the nucleosomal repeat). The finer details of histone-DNA interactions and DNA structure within these complexes is best characterized by digestion with the hydroxyl radical. Both reagents may be used to assess the degree and homogeneity of rotational and translational positioning within isolated chromatin complexes.
大多数真核细胞核内的天然染色质复合体很难通过生化方法进行研究，因此研究人员开发了研究该复合体较小部分的方法。本单元详细介绍了使用脱氧核糖核酸酶I（DNase I）和羟自由基来表征该复合体这些部分内组蛋白与DNA的相互作用。DNase I消化可用于确定DNA片段的哪些区域与核心组蛋白紧密相关，哪些区域更像裸露的DNA（即核小体重复序列内的连接DNA）。这些复合体中组蛋白与DNA相互作用及DNA结构的更精细细节，通过用羟自由基消化能得到最佳表征。这两种试剂均可用于评估分离的染色质复合体内旋转定位和平移定位的程度及均匀性。
