Carruthers L M, Bednar J, Woodcock C L, Hansen J C
Department of Biochemistry, The University of Texas Health Science Center at San Antonio 78284-7760, USA.
Biochemistry. 1998 Oct 20;37(42):14776-87. doi: 10.1021/bi981684e.
Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.
由核心组蛋白八聚体和十二段208 bp的海胆5S rDNA串联重复序列重构而成的特定核小体阵列（208 - 12核小体阵列），在MgCl₂中具有形成不稳定折叠物种的能力，其压缩程度与典型的30 nm直径高阶染色质结构相当[施瓦茨，P. M.，和汉森，J. C.（1994年）《生物化学杂志》269，16284 - 16289]。为了研究连接组蛋白在染色质凝聚中的机制功能，已将纯化的组蛋白H5与50 mM NaCl中的208 - 12核小体阵列组装在一起。随后开发了新的纯化程序，得到了208 - 12染色质模型系统的制剂，其中大部分样品每5S rDNA重复序列包含一个组蛋白八聚体，每个组蛋白八聚体包含一个组蛋白H5分子。使用分析超速离心、定量琼脂糖凝胶电泳、电子冷冻显微镜和核酸酶消化等方法，在低盐条件下对纯化的208 - 12染色质的完整性进行了广泛表征。结果表明，组蛋白H5与208 - 12核小体阵列的结合以一种产生与天然鸡红细胞染色质无法区分的结构的方式，限制了进出的连接DNA。在NaCl和MgCl₂中进行的折叠实验表明，H5的结合显著稳定了核小体阵列的中间态和广泛折叠态，而没有从根本上改变内在的核小体阵列折叠途径。这些结果通过首次证明高阶染色质结构的形成和稳定需要明显不同的大分子决定因素，为染色质折叠机制提供了新的见解。
