Dinucleosome DNA of human K562 cells: Experimental and computational characterizations
Dinucleosome DNA of human K562 cells: Experimental and computational characterizations
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DOI：
10.1016/s0022-2836(03)00838-6
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发表时间：
2003-09-05
影响因子：
5.6
通讯作者：
Kiyama, R
中科院分区：
文献类型：
--
作者：
Kato, M;Onishi, Y;Kiyama, R
二核小体的形成是组织高阶染色质结构的第一步。为了最终阐明二核小体的结构，我们构建了一个人类二核小体DNA文库。该文库由pcr扩增的DNA片段组成，这些片段是通过用微球菌核酸酶处理红系K562细胞的细胞核，然后提取DNA并将接头连接到钝端DNA片段上获得的。然后用质粒载体克隆文库并确定克隆序列。包含Alu元素的主要克隆被移除。1002个克隆组成了一个二核体数据库，其中去除Alu元素前的克隆84个，去除Alu元素后的克隆918个。约70%的克隆大小在300 ~ 400bp之间，分布在除Y染色体外的所有染色体的不同位置。根据DNA曲率图将含有A(2)N(8)A(2)N(8)A(2) A(2) A(2)或T2N8T2N8T2序列的克隆分为I型（N型）、II型（V型）和III型（M型）3种类型。实验确定的弯曲DNA片段的位置与计算的位置吻合良好，但从曲率图中可以看出，I型和ii型克隆中存在额外的弯曲DNA片段。互补二核苷酸在核小体DNA克隆末端的分布，使我们能够预测核小体二轴的位置，并估计核小体核心DNA的大小，125 nt。AA和TT二核苷酸以及其他RR和YY二核苷酸的分布呈现周期性，平均周期为10.4个碱基，与之前观察到的值接近。根据观察到的周期性，在二核小体数据库中对核小体的位置进行定位，发现核小体是由7.5+（类似于10 X n nt）的连接子分开的。这表明核小体-核小体的取向通常介于平行和反平行之间。另一个重要的发现是，AA/TT和其他RR/YY二核苷酸的分布显然反映了DNA曲率和DNA可弯曲性，共同促进了核小体的形成。(C) 2003 Elsevier Ltd.版权所有。
Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T2N8T2N8T2 sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and Ill showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125 nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ similar to 10 X n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability cooperatively contributing to the nucleosome formation. (C) 2003 Elsevier Ltd. All rights reserved.