Pif1 is a force-regulated helicase.
Pif1 is a force-regulated helicase.
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Pif1 是一种力调节解旋酶
DOI：
10.1093/nar/gkw295
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发表时间：
2016-05-19
影响因子：
14.9
通讯作者：
Dou SX
中科院分区：
文献类型：
--
作者：
Li JH;Lin WX;Zhang B;Nong DG;Ju HP;Ma JB;Xu CH;Ye FF;Xi XG;Li M;Lu Y;Dou SX
Pif 1是从细菌到人类的5′到3′ DNA解旋酶家族的典型成员。它对DNA有很高的结合亲和力，但解旋双链DNA（dsDNA）的持续合成能力很低。只有在有利于Pif 1二聚化的高蛋白浓度下才能观察到有效的DNA解旋。本研究利用单分子荧光共振能量转移（smFRET）和磁镊（MT）技术研究了酿酒酵母Pif 1（Pif 1）在不同作用力作用下的DNA解旋活性。我们发现Pif 1可以在零力下重复地解开分叉的DNA许多解旋-重新压缩循环。然而，发现Pif 1在每个循环中具有非常有限的持续合成能力，因为它很容易放松其与跟踪链的强关联，这解释了为什么在低蛋白浓度下在批量测定中不能观察到Pif 1有效地解旋DNA。该力显著提高了Pif 1的解旋速率和总解旋长度。在9 pN的力下，速率和长度分别增加了3倍和20倍以上。我们的研究结果表明，Pif 1的DNA解旋活性可以通过力来调节。PIF 1的这种特性与其细胞功能的相关性进行了讨论。
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.