Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.
PLoS One. 2012;7(9):e46306. doi: 10.1371/journal.pone.0046306. Epub 2012 Sep 25.
While many proteins are involved in the assembly and (re)positioning of nucleosomes, the dynamics of protein-assisted nucleosome formation are not well understood. We study NAP1 (nucleosome assembly protein 1) assisted nucleosome formation at the single-molecule level using magnetic tweezers. This method allows to apply a well-defined stretching force and supercoiling density to a single DNA molecule, and to study in real time the change in linking number, stiffness and length of the DNA during nucleosome formation. We observe a decrease in end-to-end length when NAP1 and core histones (CH) are added to the dsDNA. We characterize the formation of complete nucleosomes by measuring the change in linking number of DNA, which is induced by the NAP1-assisted nucleosome assembly, and which does not occur for non-nucleosomal bound histones H3 and H4. By rotating the magnets, the supercoils formed upon nucleosome assembly are removed and the number of assembled nucleosomes can be counted. We find that the compaction of DNA at low force is about 56 nm per assembled nucleosome. The number of compaction steps and associated change in linking number indicate that NAP1-assisted nucleosome assembly is a two-step process.
虽然许多蛋白质参与核小体的组装和（再）定位，但蛋白质辅助核小体形成的动力学还不是很清楚。我们使用磁镊在单分子水平上研究 NAP1（核小体组装蛋白 1）辅助核小体的形成。这种方法可以对单个 DNA 分子施加定义明确的拉伸力和超螺旋密度，并实时研究核小体形成过程中 DNA 的连接数、刚性和长度的变化。当 NAP1 和核心组蛋白（CH）被添加到 dsDNA 中时，我们观察到末端到末端长度的减少。我们通过测量由 NAP1 辅助核小体组装诱导的 DNA 的连接数的变化来表征完整核小体的形成，而对于非核小体结合的组蛋白 H3 和 H4 则不会发生这种变化。通过旋转磁铁，可以去除核小体组装时形成的超螺旋，并且可以计数组装的核小体的数量。我们发现，在低力下 DNA 的压缩约为每个组装的核小体 56nm。压缩步骤的数量和相关的连接数变化表明，NAP1 辅助核小体的组装是一个两步过程。
