The WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium &ITThermotoga elfii&IT is an Accessory Single-Stranded DNA Binding Module
The WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium &ITThermotoga elfii&IT is an Accessory Single-Stranded DNA Binding Module
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DOI：
10.1021/acs.biochem.7b01233
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发表时间：
2018-02-20
期刊：
影响因子：
2.9
通讯作者：
Bochman, Matthew L.
中科院分区：
文献类型：
--
作者：
Andis, Nicholas M.;Sausen, Christopher W.;Bochman, Matthew L.
PIF1家族解旋酶从细菌到人类都是保守的。然而，除了研究得很好的酵母PIF1解旋酶(如ScPif1和ScRrm3)外，人们对这些酶如何帮助维持基因组稳定知之甚少。事实上，我们对这些蛋白质中特有的中央PIF1解旋酶结构域的N-末端和C-末端缺乏基本的了解。在这里，使用嵌合构建物，我们证明了ScPif1和ScRrm3解旋酶结构域是可互换的，并且ScRrm3的N末端对其在体内的功能是重要的。这表明，PIF1家族解旋酶进化出融合到通用运动域的功能模块。为了验证这一假设，我们对嗜热菌Thermotoga elfii(TePif1)的PIF1解旋酶(TePif1)的生化活性进行了表征，该酶含有一个功能未知的C端WYL结构域。与其他嗜热菌的解旋酶一样，重组TePif1制备简单，体外热稳定性好，显示出与其真核同源物相似的活性。我们还发现，WYL结构域是高亲和力单链DNA(SsDNA)结合所必需的，并同时影响ATPase和解旋酶的活性。从TePif1中删除WYL结构域或突变预测的ssDNA结合位点上的保守残基使ATPase活性和DNA解离，导致ATP水解率较高，但DNA解旋酶活性较低。我们的发现表明，在真核生物PIF1解旋酶中发现的未知功能域也可能赋予这些酶功能特异性和额外的活性，这应该在未来的工作中进行研究。
PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function in vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable in vitro, and displayed activities similar to its eukaryotic homologues. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work.