Li Jing-Hua, Lin Wen-Xia, Zhang Bo, Nong Da-Guan, Ju Hai-Peng, Ma Jian-Bing, Xu Chun-Hua, Ye Fang-Fu, Xi Xu Guang, Li Ming, Lu Ying, Dou Shuo-Xing
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.
College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China.
Nucleic Acids Res. 2016 May 19;44(9):4330-9. doi: 10.1093/nar/gkw295. Epub 2016 Apr 20.
Pif1 is a prototypical member of the 5' to 3' DNA helicase family conserved from bacteria to human. It has a high binding affinity for DNA, but unwinds double-stranded DNA (dsDNA) with a low processivity. Efficient DNA unwinding has been observed only at high protein concentrations that favor dimerization of Pif1. In this research, we used single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) to study the DNA unwinding activity of Saccharomyces cerevisiae Pif1 (Pif1) under different forces exerted on the tails of a forked dsDNA. We found that Pif1 can unwind the forked DNA repetitively for many unwinding-rezipping cycles at zero force. However, Pif1 was found to have a very limited processivity in each cycle because it loosened its strong association with the tracking strand readily, which explains why Pif1 cannot be observed to unwind DNA efficiently in bulk assays at low protein concentrations. The force enhanced the unwinding rate and the total unwinding length of Pif1 significantly. With a force of 9 pN, the rate and length were enhanced by more than 3- and 20-fold, respectively. Our results imply that the DNA unwinding activity of Pif1 can be regulated by force. The relevance of this characteristic of Pif1 to its cellular functions is discussed.
Pif1是从细菌到人类保守的5'至3'DNA解旋酶家族的典型成员。它对DNA具有高结合亲和力，但以低持续性解旋双链DNA（dsDNA）。仅在有利于Pif1二聚化的高蛋白浓度下才观察到有效的DNA解旋。在本研究中，我们使用单分子荧光共振能量转移（smFRET）和磁镊（MT）来研究酿酒酵母Pif1（Pif1）在施加于叉状dsDNA尾部的不同力作用下的DNA解旋活性。我们发现，Pif1可以在零力下重复解旋叉状DNA许多解旋-重新拉链循环。然而，发现Pif1在每个循环中的持续性非常有限，因为它很容易松开与追踪链的强结合，这解释了为什么在低蛋白浓度的大量测定中无法观察到Pif1有效地解旋DNA。力显著提高了Pif1的解旋速率和总解旋长度。在9 pN的力作用下，速率和长度分别提高了3倍和20倍以上。我们的结果表明，Pif1的DNA解旋活性可以受到力的调节。讨论了Pif1这一特性与其细胞功能的相关性。
