Visualizing single DNA-bound proteins using DNA as a scanning probe
Visualizing single DNA-bound proteins using DNA as a scanning probe
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DOI：
10.1038/nmeth1126
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发表时间：
2007-12-01
期刊：
影响因子：
48
通讯作者：
Wuite, Gijs J. L.
中科院分区：
文献类型：
--
作者：
Noom, Maarten C.;van den Broek, Bram;Wuite, Gijs J. L.
许多生物过程都涉及酶沿着DNA移动。这种运动可能会受到DNA结合蛋白或DNA超级线圈的阻碍。目前的技术无法直接测量这种“路障”可能施加的力量。我们用四个独立移动的光学陷阱构建了一个装置，使我们能够操纵夹在珠子之间的两个DNA分子。通过将一个DNA紧紧包裹在另一个DNA上，我们创造了一个探针，可以沿着第二个DNA的轮廓扫描。我们发现两种聚合物之间的摩擦保持在1pn以下。在遇到dna结合的蛋白质时，测量大量的摩擦力，从而精确定位蛋白质的位置。此外，这些蛋白质在低探针张力下保持相关，但可以在大于20 pN的力下被驱赶。最后，完全控制两个DNA分子的方向开启了与多个DNA区域相互作用的蛋白质的广泛实验。
Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.