A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End.
A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End.
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DOI：
10.1016/j.jmb.2016.02.017
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发表时间：
2016-03-27
影响因子：
5.6
通讯作者：
Galletto R
中科院分区：
文献类型：
--
作者：
Singh SP;Koc KN;Stodola JL;Galletto R
使用DNA聚合酶偶联分析和基于FRET的解旋酶分析，在这项工作中，我们证明了酿酒酵母Pif1的一个单体可以解开双链DNA。Pif1单体的解旋酶活性受底物3‘-ssDNA尾部的性质及其对依赖于Pif1的卷绕活性的影响，该卷曲活性与dsDNA的打开相耦合。我们认为，除了与转运链相互作用的蛋白质上的单链DNA位点外，Pif1还有第二个结合底物3‘-单链DNA的位点。DNA与这个位点的相互作用调节了回绕抵消解旋的程度。根据3‘-尾的性质和要解开的双链DNA的长度，这种活性足以掩盖单体的解旋酶活性。在Pif1超过DNA的情况下，依赖于Pif1的开放DNA的回绕强烈地限制了解离，独立于3‘-尾巴。我们认为，在这种情况下，DNA与第二个位点的结合被排除，并且依赖于Pif1的卷绕活性的调制在很大程度上丢失了。
Using a DNA polymerase-coupled assay and FRET-based helicase assays, in this work we show that a monomer of S. cerevisiae Pif1 can unwind double-stranded DNA. The helicase activity of a Pif1 monomer is modulated by the nature of the 3′-ssDNA tail of the substrate and its effect on a Pif1-dependent re-winding activity that is coupled to the opening of dsDNA. We propose that in addition to the ssDNA site on the protein that interacts with the translocating strand, Pif1 has a second site that binds the 3′-ssDNA of the substrate. Interaction of DNA with this site modulates the degree to which re-winding counteracts unwinding. Depending on the nature of the 3′-tail and the length of the duplex DNA to be unwound this activity is sufficiently strong to mask the helicase activity of a monomer. In excess Pif1 over the DNA the Pif1-dependent re-winding of the opened DNA strongly limits unwinding, independent of the 3′-tail. We propose that in this case binding of DNA to the second site is precluded and modulation of the Pif1-dependent re-winding activity is largely lost.