The hexametric T7 helicase (gp4) adopts a spiral lock-washer form and encircles a coil-like DNA (tracking) strand with two nucleotides bound to each subunit. However, the chemo-mechanical coupling mechanism in unwinding has yet to be elucidated. Here, we utilized nanotensioner-enhanced Förster resonance energy transfer with one nucleotide precision to investigate gp4-induced unwinding of DNA that contains an abasic lesion. We observed that the DNA unwinding activity of gp4 is hindered but not completely blocked by abasic lesions. Gp4 moves back and forth repeatedly when it encounters an abasic lesion, whereas it steps back only occasionally when it unwinds normal DNA. We further observed that gp4 translocates on the tracking strand in step sizes of one to four nucleotides. We propose that a hypothetical intermediate conformation of the gp4-DNA complex during DNA unwinding can help explain how gp4 molecules pass lesions, providing insights into the unwinding dynamics of gp4.

中文翻译：

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通过噬菌体T7解旋酶解开DNA分子机制的动态结构见解。

六边形T7解旋酶（gp4）采用螺旋锁紧垫圈形式，并围绕一个盘绕状DNA（跟踪）链，每个亚基结合了两个核苷酸。但是，放卷中的化学机械耦合机理尚未阐明。在这里，我们利用具有一个核苷酸精度的纳米张紧器增强的Förster共振能量转移来研究gp4诱导的包含无碱基病变的DNA的解旋。我们观察到，gp4的DNA解旋活性受基础性病变的阻碍，但并未完全被阻止。Gp4遇到无基础性病变时会反复地来回移动，而当它解开正常的DNA时它只会偶尔后退。我们进一步观察到，gp4以1至4个核苷酸的步长在跟踪链上移位。