Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation
Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation
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DOI：
10.1128/mcb.22.20.7147-7157.2002
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发表时间：
2002-10-01
影响因子：
5.3
通讯作者：
Widom, J
中科院分区：
文献类型：
--
作者：
Anderson, JD;Thåström, A;Widom, J
被称为“位点暴露”的内在核小体动力学提供了对体外核小体 DNA 埋藏区域的自发且合作的访问。已经提出了两种不同的位点暴露机制，一种基于核小体易位，另一种基于动态核小体构象变化，其中一段核小体 DNA 短暂地从组蛋白表面释放。在这里，我们报告了区分这些机制的三个实验。一项实验研究了当额外的 DNA（核小体可以以低能量成本移动到其上）被附加到一端时，对核小体内限制性内切酶靶位点的可及性的影响。另外两个实验在用于探测限制酶可及性的条件下直接测试核小体迁移率：一个在选定的非天然核小体定位序列上，另一个在经过充分研究的 5S rRNA 基因核小体定位序列上。我们从所有三个测定中发现，限制性内切酶可以进入核小体 DNA 整个长度的位点，而不受核小体易位的影响。我们得出的结论是，体外核小体中的位点暴露是通过核小体构象变化发生的，该变化导致一段DNA从组蛋白表面短暂释放，很可能涉及从末端逐渐解卷。在部分解卷的 DNA 远端位点重新捕获将产生环状结构，据信该结构有助于 RNA 聚合酶延伸，并可能有助于自发或 ATP 驱动的核小体迁移。短暂的开放状态可能有利于转录因子和酶最初进入体内。
Intrinsic nucleosome dynamics termed "site exposure" provides spontaneous and cooperative access to buried regions of nucleosomal DNA in vitro. Two different mechanisms for site exposure have been proposed, one based on nucleosome translocation, the other on dynamic nucleosome conformational changes in which a stretch of the nucleosomal DNA is transiently released off the histone surface. Here we report on three experiments that distinguish between these mechanisms. One experiment investigates the effects on the accessibilities of restriction enzyme target sites inside nucleosomes when extra DNA (onto which the nucleosome may move at low energetic cost) is appended onto one end. The other two experiments test directly for nucleosome mobility under the conditions used to probe accessibility to restriction enzymes: one on a selected nonnatural nucleosome positioning sequence, the other on the well-studied 5S rRNA gene nucleosome positioning sequence. We find from all three assays that restriction enzymes gain access to sites throughout the entire length of the nucleosomal DNA without contribution from nucleosome translocation. We conclude that site exposure in nucleosomes in vitro occurs via a nucleosome conformational change that leads to transient release of a stretch of DNA from the histone surface, most likely involving progressive uncoiling from an end. Recapture at a distal site along DNA that has partially uncoiled would result in looped structures which are believed to contribute to RNA polymerase elongation and may contribute to spontaneous or ATP-driven nucleosome mobility. Transient open states may facilitate the initial entry of transcription factors and enzymes in vivo.