Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA.
Biophys J. 2009 Dec 16;97(12):3150-7. doi: 10.1016/j.bpj.2009.09.032.
We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers. To verify our hypothesis that the lack of full nucleosome assembly on topologically constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we carried out experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Indeed, such rotation did lead to the same nucleosome saturation level as in the case of nicked tethers. We conclude that levels of positive supercoiling in the range of 0.025-0.051 (most probably in the form of twist) stall the nucleosome assembly process.
我们使用磁镊研究拓扑约束 DNA 分子上核小体的组装。在恒定的低力下，使用鸡红细胞核心组蛋白和组蛋白伴侣蛋白 Nap1 实现组装。当 DNA 受到拓扑约束时，我们只观察到部分组装，而在不受约束（缺口）的 DNA 系链上则观察到更完全的组装。为了验证我们的假设，即拓扑约束系链上没有完全的核小体组装是由于模板其余部分的正超螺旋的补偿性积累，我们进行了实验，在这些实验中，我们通过在组装过程的某些时间点旋转外部磁场来机械地缓解正超螺旋。事实上，这种旋转确实导致了与缺口系链相同的核小体饱和水平。我们得出结论，正超螺旋的水平在 0.025-0.051 之间（很可能以扭曲的形式）会阻碍核小体组装过程。
