Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding
Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding
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DOI：
10.1074/jbc.m117.814178
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发表时间：
2017-11-17
影响因子：
4.8
通讯作者：
Trakselis, Michael A.
中科院分区：
文献类型：
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作者：
Carney, Sean M.;Gomathinayagam, Shivasankari;Trakselis, Michael A.
复制六聚体解旋酶被认为是通过空间排斥（SE）解除双链DNA，其中一条DNA链被六聚体包围，另一条被排除在中心通道之外。然而，与六聚解旋酶外表面的排除链的相互作用也被证明对DNA解绕很重要，从而产生了空间排除和包裹（SEW）模型。例如，古细菌Sulfolobus solfataricus迷你染色体维持（SsoMCM）解旋酶已被证明通过SEW模式解绕DNA以提高解绕效率。使用单分子FRET，我们现在发现类似的大肠杆菌（Ec） DNA解旋酶在解绕过程中也与排除的DNA链特异性相互作用。与野生型相比，EcDnaB外表面的几个保守和带正电的残基突变导致相互作用的动态和状态增加。令人惊讶的是，这些突变还增加了DNA的解绕率，这表明与被排除的链的静电接触可以作为解绕活性的调节器。为了支持这一点，用morpholino底物而不是DNA中和被排除链的电荷的实验也显著提高了解绕速率。值得注意的是，尽管EcDnaB和SsoMCM的排除链的稳定性几乎相同，但这些酶来自不同的超家族，并以相反的极性解开DNA。这些结果支持EcDnaB解绕的SEW模型，该模型扩展了现有的六聚解旋酶解绕的SE模型，包括被排除链调节DNA解绕速率的贡献。
Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal Sulfolobus solfataricus minichromosome maintenance (SsoMCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous Escherichia coli (Ec) DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively charged residues on the exterior surface of EcDnaB resulted in increased interaction dynamics and states compared with wild type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical for EcDnaB and SsoMCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding for EcDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.