Wagner Gaudeline, Bancaud Aurélien, Quivy Jean-Pierre, Clapier Cédric, Almouzni Geneviève, Viovy Jean-Louis
Laboratoire PhysicoChimie Curie, Institut Curie, CNRS UMR 168, 75248 Paris, France.
Biophys J. 2005 Nov;89(5):3647-59. doi: 10.1529/biophysj.105.062786. Epub 2005 Aug 12.
Kinetics of compaction on single DNA molecules are studied by fluorescence videomicroscopy in the presence of 1), Xenopus egg extracts and 2), purified nucleosome reconstitution systems using a combination of histones with either the histone chaperone Nucleosome Assembly Protein (NAP-1) or negatively charged macromolecules such as polyglutamic acid and RNA. The comparison shows that the compaction rates can differ by a factor of up to 1000 for the same amount of histones, depending on the system used and on the presence of histone tails, which can be subjected to post-translational modifications. Reactions with purified reconstitution systems follow a slow and sequential mechanism, compatible with the deposition of one (H3-H4)(2) tetramer followed by two (H2A-H2B) dimers. Addition of the histone chaperone NAP-1 increases both the rate of the reaction and the packing ratio of the final product. These stimulatory effects cannot be obtained with polyglutamic acid or RNA, suggesting that yNAP-1 impact on the reaction cannot simply be explained in terms of charge screening. Faster compaction kinetics and higher packing ratios are reproducibly reached with extracts, indicating a role of additional components present in this system. Data are discussed and models proposed to account for the kinetics obtained in our single-molecule assay.
在以下两种情况下，通过荧光视频显微镜研究单个DNA分子的压缩动力学：1）非洲爪蟾卵提取物；2）使用组蛋白与组蛋白伴侣核小体组装蛋白（NAP-1）或带负电荷的大分子（如聚谷氨酸和RNA）组合的纯化核小体重构系统。比较结果表明，对于相同数量的组蛋白，压缩速率可能相差高达1000倍，这取决于所使用的系统以及组蛋白尾巴的存在情况，组蛋白尾巴可进行翻译后修饰。与纯化的重构系统的反应遵循缓慢且有序的机制，这与先沉积一个（H3-H4）(2) 四聚体，然后再沉积两个（H2A-H2B）二聚体的过程相符。添加组蛋白伴侣NAP-1会提高反应速率和最终产物的堆积率。聚谷氨酸或RNA无法产生这些刺激作用，这表明yNAP-1对反应的影响不能简单地用电荷屏蔽来解释。提取物可重复性地实现更快的压缩动力学和更高的堆积率，这表明该系统中存在的其他成分发挥了作用。我们对数据进行了讨论，并提出了模型来解释在单分子检测中获得的动力学。
