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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Nucleosome disassembly intermediates characterized by single-molecule FRET
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Nucleosome disassembly intermediates characterized by single-molecule FRET

机译：以单分子FRET为特征的核小体拆卸中间体

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摘要

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated "Widom 601" positioning sequence labeled with donor and acceptor flu-orophores. Reversible dissociation was induced by increasing NaCI concentration. At least 3 species with different FRET were identified and assigned to structures: (ⅰ) the most stable high-FRET species corresponding to the intact nucleosome, (ⅱ) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (ⅲ) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 A measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.
机译：核小体在基因组DNA的紧缩和DNA转录和复制可及性的控制中起着核心作用。为了帮助理解这些过程中核小体打开和关闭的机制，我们使用了带有供体和受体fluorophores标记的SELEX生成的“ Widom 601”定位序列，通过具有高空间分辨率的定量单分子FRET研究了单核小体的拆卸。通过增加NaCl浓度诱导可逆解离。鉴定出至少3种具有不同FRET的物种并将其分配给以下结构：（ⅰ）对应完整核小体的最稳定的高FRET物种；（ⅱ）我们认为归因于第一中间体的部分稳定性较低的中等FRET物种（F）一种低FRET物种，其特征是非常广泛的FRET分布，代表高度解开的结构，并且形成了游离DNA，而其他2种却以其为代价。选择性FCS分析表明，即使在低FRET状态下，某些组蛋白仍与DNA结合。对于高FRET物质测得的染料间距离为54.0 A，对应于接近已知晶体结构的紧密构象。这些物种的共存和相互转化首先在非侵入性条件下得到证明。 DNA展开的几何模型可预测观察到的FRET物种的存在。这些物种在分解途径中的不同结构绘制了能量分布图，表明10 bp的主要障碍和5 bp DNA展开步骤的次要障碍。

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    Alexander Gansen; Alessandro Valeri; Florian Hauger; Suren Felekyan; Stanislav Kalinin; Katalin Toth; Joerg Langowski; Claus A. M. Seidel;

  • 作者单位

    Abteilung Biophysik der Makromolekuele, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany;

    Lehrstuhl fuer Molekulare Physikalische Chemie, Heinrich-Heine-Universitaet, Universitaetsstrasse 1, Geb. 26.32, 40225 Duesseldorf, Germany;

    Abteilung Biophysik der Makromolekuele, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany;

    Lehrstuhl fuer Molekulare Physikalische Chemie, Heinrich-Heine-Universitaet, Universitaetsstrasse 1, Geb. 26.32, 40225 Duesseldorf, Germany;

    Lehrstuhl fuer Molekulare Physikalische Chemie, Heinrich-Heine-Universitaet, Universitaetsstrasse 1, Geb. 26.32, 40225 Duesseldorf, Germany;

    Abteilung Biophysik der Makromolekuele, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany;

    Abteilung Biophysik der Makromolekuele, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany;

    Lehrstuhl fuer Molekulare Physikalische Chemie, Heinrich-Heine-Universitaet, Universitaetsstrasse 1, Geb. 26.32, 40225 Duesseldorf, Germany;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    chromatin; mononucieosomes; structure;

    机译：染色质单核小体;结构体;

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