Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking
Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking
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DOI：
10.1038/s41598-020-70763-y
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发表时间：
2020-08-13
影响因子：
4.6
通讯作者：
Kamagata，Kiyoto
中科院分区：
文献类型：
--
作者：
Subekti，Dwiky Rendra Graha;Murata，Agato;Kamagata，Kiyoto
表征的目标搜索动力学的DNA结合蛋白沿着DNA已受到阻碍的时间分辨率的标准单分子荧光显微镜。在这里，我们实现了0.5 ms的时间分辨率在荧光显微镜测量通过优化的荧光激发的基础上的临界角照明，并通过利用电子倍增电荷耦合器件的时间延迟积分模式。我们的特点是肿瘤抑制基因p53沿着非特异性DNA在生理盐浓度的目标搜索动力学。我们确定了一个短暂的遭遇中间形成的长寿命的p53-DNA复合物。在较高的盐浓度下，p53沿着DNA的跳跃和一维扩散都被加速，这表明p53沿着DNA凹槽的旋转解偶联运动和p53/DNA复合物的构象变化。这种方法可以用来澄清以前隐藏的时间平均的DNA结合蛋白的未解决的动态。
Characterization of the target search dynamics of DNA-binding proteins along DNA has been hampered by the time resolution of a standard single-molecule fluorescence microscopy. Here, we achieved the time resolution of 0.5 ms in the fluorescence microscopy measurements by optimizing the fluorescence excitation based on critical angle illumination and by utilizing the time delay integration mode of the electron-multiplying charge coupled device. We characterized the target search dynamics of the tumor suppressor p53 along nonspecific DNA at physiological salt concentrations. We identified a short-lived encounter intermediate before the formation of the long-lived p53–DNA complex. Both the jumps and the one-dimensional diffusion of p53 along DNA were accelerated at higher salt concentrations, suggesting the rotation-uncoupled movement of p53 along DNA grooves and conformational changes in the p53/DNA complex. This method can be used to clarify the unresolved dynamics of DNA-binding proteins previously hidden by time averaging.