Fitzgerald D J, Anderson J N
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
J Biol Chem. 1999 Sep 17;274(38):27128-38. doi: 10.1074/jbc.274.38.27128.
Previous studies have shown that drugs which bind in the DNA minor groove reduce the curvature of bent DNA. In this article, we examined the effects of these drugs on the nucleosome assembly of DNA molecules that display different degrees of intrinsic curvature. DAPI (4,6-diamidino-2-phenylindole) inhibited the assembly of a histone octamer onto a 192-base pair circular DNA fragment from Caenorhabditis elegans and destabilized a nucleosome that was previously assembled on this segment. The inhibitory effect was highly selective since it was not seen with nonbent molecules, bent molecules with noncircular shapes, or total genomic DNA. This marked template specificity was attributed to the binding of the ligand to multiple oligo A-tracts distributed over the length of the fragment. A likely mechanism for the effect is that the bound ligand prevents the further compression of the DNA into the minor groove which is required for assembly of DNA into nucleosomes. To further characterize the effects of the drug on chromatin formation, a nucleosome was assembled onto a 322-base pair DNA fragment that contained the circular element and a flanking nonbent segment of DNA. The position of the nucleosome along the fragment was then determined using a variety of nuclease probes including exonuclease III, micrococcal nuclease, DNase I, and restriction enzymes. The results of these studies revealed that the nucleosome was preferentially positioned along the circular element in the absence of DAPI but assembled onto the nonbent flanking sequence in the presence of the drug. DAPI also induced the directional movement of the nucleosome from the circular element onto the nonbent flanking sequence when a nucleosome preassembled onto this template was exposed to the drug under physiologically relevant conditions.
先前的研究表明，结合于DNA小沟的药物会降低弯曲DNA的曲率。在本文中，我们研究了这些药物对呈现不同程度固有曲率的DNA分子核小体组装的影响。4,6-二脒基-2-苯基吲哚（DAPI）抑制组蛋白八聚体组装到秀丽隐杆线虫的192个碱基对的环状DNA片段上，并使先前组装在该片段上的核小体不稳定。这种抑制作用具有高度选择性，因为在非弯曲分子、非圆形的弯曲分子或总基因组DNA中未观察到这种作用。这种显著的模板特异性归因于配体与分布在片段长度上的多个寡聚A序列的结合。这种作用的一个可能机制是，结合的配体阻止了DNA进一步压缩到小沟中，而这是DNA组装成核小体所必需的。为了进一步表征该药物对染色质形成的影响，将一个核小体组装到一个322个碱基对的DNA片段上，该片段包含环状元件和侧翼的非弯曲DNA片段。然后使用包括外切核酸酶III、微球菌核酸酶、DNase I和限制酶在内的多种核酸酶探针来确定核小体在片段上的位置。这些研究结果表明，在没有DAPI的情况下，核小体优先定位在环状元件上，但在有药物存在时则组装到非弯曲的侧翼序列上。当预先组装在该模板上的核小体在生理相关条件下暴露于药物时，DAPI还诱导核小体从环状元件向非弯曲的侧翼序列定向移动。
