Lin Wenxia, Ma Jianbing, Nong Daguan, Xu Chunhua, Zhang Bo, Li Jinghua, Jia Qi, Dou Shuoxing, Ye Fangfu, Xi Xuguang, Lu Ying, Li Ming
Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.
School of Physical Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
Phys Rev Lett. 2017 Sep 29;119(13):138102. doi: 10.1103/PhysRevLett.119.138102. Epub 2017 Sep 26.
Single-molecule Förster resonance energy transfer is widely applied to study helicases by detecting distance changes between a pair of dyes anchored to overhangs of a forked DNA. However, it has been lacking single-base pair (1-bp) resolution required for revealing stepping kinetics of helicases. We designed a nanotensioner in which a short DNA is bent to exert force on the overhangs, just as in optical or magnetic tweezers. The strategy improved the resolution of Förster resonance energy transfer to 0.5 bp, high enough to uncover differences in DNA unwinding by yeast Pif1 and E. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1 bp at a time but sequesters the nascent nucleotides and releases them randomly. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.
单分子荧光共振能量转移通过检测附着在叉状DNA突出端的一对染料之间的距离变化，被广泛应用于解旋酶的研究。然而，它一直缺乏揭示解旋酶步进动力学所需的单碱基对（1-bp）分辨率。我们设计了一种纳米张力器，其中一段短DNA被弯曲以对突出端施加力，就像在光学或磁镊中一样。该策略将荧光共振能量转移的分辨率提高到了0.5 bp，这一分辨率足以揭示酵母Pif1和大肠杆菌RecQ解旋DNA的差异，而目前的方法无法区分它们的解旋行为。我们发现，Pif1表现出1-bp步进动力学，而RecQ一次解开1 bp，但会隔离新生核苷酸并随机释放它们。高分辨率数据使我们能够提出一个三参数模型，以定量解释这两种属于两个超家族的解旋酶明显不同的解旋行为。
