Kato Megumi, Onishi Yoshiaki, Wada-Kiyama Yuko, Kiyama Ryoiti
Research Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
J Mol Biol. 2005 Jul 8;350(2):215-27. doi: 10.1016/j.jmb.2005.04.075.
The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.
双核小体是用于分析高阶染色质结构的信息单元。在体外重新组装核小体并用微球菌核酸酶消化后，从双核小体DNA文库中筛选出形成稳定双核小体的DNA片段。重组的双核小体对微球菌核酸酶表现出不同的敏感性，这表明双核小体的生化稳定性部分取决于DNA片段。筛选后的DNA片段根据对微球菌核酸酶的敏感性分为以克隆bf10、af14和af32为代表的三组。通过限制性消化后的DNA的Southern印迹和引物延伸分析对核小体边界进行定位，结果表明克隆af32的每个核小体位置都是固定的。使用克隆af32的突变DNA片段对重组双核小体进行分析，揭示了关键核小体的独特性质，因为对应于右核小体位置的DNA片段的替换导致对微球菌核酸酶的敏感性显著增加，而与原始af32构建体相比，其他核小体片段的替换对敏感性几乎没有影响。去除右核小体的突变构建体显示出多个核小体阶段，这表明右核小体首先稳定每个单核小体，然后稳定双核小体。寡核苷酸弯曲试验表明，右核小体中的DNA片段包含弯曲的DNA，这表明核小体的定位活性归因于其DNA结构。这些结果表明，形成稳定双核小体的信息嵌入在基因组DNA中，对关键核小体的进一步表征有助于理解染色质结构的构建。
