Nucleosome Structure-Function Relationship : Analyses by using atomic force microscopy

核小体结构-功能关系：使用原子力显微镜进行分析

基本信息

批准号：
10480194
负责人：
TAKEYASU Kunio
金额：
$ 7.04万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

We have previously developed the analytical techniques of atomic force microscopy (AFM) for the study of interaction between DNA and DNA-binding proteins. Here we further extended the AFM application to the higher-order structures of DNA/protein complexes.(1) The DNase I-hyper-sensitive sites (HS2-HS4) in the β-globin gene enhancer region (locus control region ; LCR) were found to form a looped-DNA structure as the target of Bach1/MafK heterodimers. Further detailed analyses of the loop formation proposed a novel 'kiss and pull' model for the enhancer/protein interaction : the Bach1/MafK heterodimer preferentially binds to HS2 with highest affinity and to HS3 with lower affinity, resulting in a stay and leave at the HS2 and HS3 sites, respectively, by forming a stable complex of 4 heterodimers (J.Electron Microscopy, 49 : 407-413).(2) The resolutions of AFM have been limited to an inherent property of the technique ; tip effect associated with a large radius of the scanning probe. To o … More vercome this problem, we developed a carbon nanotube probe by attaching a carbon nanotube to a conventional scanning probe under a well-controlled process. Because of the constant and small radius of the tip (2.5-10nm) and the high aspect ratio (1 : 100) of carbon nanotube, the lateral resolutions have been much improved, and enabled us to clearly visualize the subunit organization of multi-subunit proteins (J.Electron Microscopy, 49 : 415-421 ; Proc.Nat'l Acad.Sci.USA, 97 : 14127-14132).(3) DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication and recombination. When a replication initiator protein, RepE54, binds to the specific sequences (iterons) of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state without a DNA strand beak, a local melting, nor a DNA wrapping. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state (Biochemistry, 39 : 9139-9145).(4) To address the question of how nuclear histones and DNA interact and form a nucleosome structure, an in vitro reconstituted chromatin system was established in which AFM can be applied to the visualization of a "beads-on-a-string" structure with each nucleosome trapping 150 bp DNA and constant spacing. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure (FEBS lett., ). A Substitution of H3 for a centromere specific protein CENP-A resulted in the formation of centromere-specific nucleosomes with a well-known beads-on-a-string structure consisted of only about 110bp DNA.Moreover, the volume of centromere-specific nucleosome was significantly smaller (15%) than that of control nucleosome (Proc.Nat'l Acad.Sci.USA, 97 : 7266-7271). Less

我们之前开发了原子力显微镜 (AFM) 分析技术，用于研究 DNA 和 DNA 结合蛋白之间的相互作用。在这里，我们进一步将 AFM 应用扩展到 DNA/蛋白质复合物的高阶结构。(1) β-珠蛋白基因增强子区（位点控制区；LCR）中的 DNase I 超敏感位点 (HS2-HS4) 被发现形成环状 DNA 结构，作为 Bach1/MafK 的靶标对环形成的进一步详细分析提出了一种新的增强子/蛋白质相互作用的“亲吻和拉动”模型：Bach1/MafK 异二聚体优先与具有最高亲和力的 HS2 结合，并与具有较低亲和力的 HS3 结合，从而导致停留和离开。分别在 HS2 和 HS3 位点，通过形成 4 个异二聚体的稳定复合物（J.Electron Microscopy，49： 407-413).(2) AFM 的分辨率受限于该技术的固有特性；与大半径扫描探针相关的尖端效应。为了解决这个问题，我们开发了一种碳纳米管探针。在良好控制的过程下将碳纳米管附着到传统的扫描探针上，因为碳纳米管的尖端半径恒定且小（2.5-10nm），并且碳纳米管具有高纵横比（1：100）。横向分辨率大大提高，使我们能够清楚地可视化多亚基蛋白质的亚基组织（J.Electron Microscopy，49：415-421；Proc.Nat'l Acad.Sci.USA，97：14127-14132 (3)已知DNA超螺旋在多种细胞事件中发挥着重要作用，例如转录、复制和重组。 RepE54 蛋白与负超螺旋 mini-F 质粒的特定序列（迭代子）结合，诱导质粒动态结构转变为松弛状态，没有 DNA 链喙、局部解链或 DNA 包裹数据。表明由引发剂结合施加的局部应变可以诱导 DNA 构象从超螺旋状态急剧转变为松弛状态（生物化学，39： 9139-9145）。（4）为了解决核组蛋白和DNA如何相互作用并形成核小体结构的问题，建立了体外重构染色质系统，其中AFM可以应用于“珠子上的珠子”的可视化每个核小体捕获 150 bp DNA 且间距恒定的“-string”结构向系统中添加组蛋白 H1 导致紧密压缩。双核小体结构（FEBS lett.，A）用 H3 替换着丝粒特异性蛋白 CENP-A，导致形成着丝粒特异性核小体，其具有众所周知的仅由约 110bp DNA 组成的串珠结构。此外，着丝粒特异性核小体的体积明显小于对照核小体的体积（15%）（美国国家科学院院刊，97：7266-7271）。