Conformational dynamics of a Y-family DNA polymerase during substrate binding and catalysis as revealed by interdomain Förster resonance energy transfer.
Conformational dynamics of a Y-family DNA polymerase during substrate binding and catalysis as revealed by interdomain Förster resonance energy transfer.
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DOI：
10.1021/bi5000146
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发表时间：
2014-03-25
期刊：
影响因子：
2.9
通讯作者：
Suo Z
中科院分区：
文献类型：
--
作者：
Maxwell BA;Xu C;Suo Z
许多动力学、结构和理论研究已经确定，DNA聚合酶调整其结构域结构以将核苷酸封闭在其活性位点中，然后重新排列关键活性位点残基和催化底物，后者的构象变化在动力学上限制正确的核苷酸掺入速率。此外，结构研究已经揭示了Y家族DNA聚合酶的脱辅基蛋白和DNA-蛋白质二元状态之间的大的构象变化。在以前的研究中[Xu，C.，麦克斯韦，B。一、布朗，J.A.，张，L.，和Suo，Z. 7，e1000225]，开发了实时Förster共振能量转移（FRET）方法，以通过测量酶和DNA底物上的位置之间的距离变化来监测核苷酸结合和掺入期间来自硫磺硫化叶菌（Sulfolobus solfataricus）（Dpo 4）的DNA聚合酶IV（原型Y家族酶）的全局构象转变。为了阐明底物结合和催化过程中Dpo 4构象转变的进一步细节，在这项研究中，实时FRET技术被用来监测蛋白质本身中各种位置对之间的距离变化。除了提供新的见解的构象变化揭示在以前的研究中，这里的结果表明，先前描述的DPO 4的载脂蛋白和DNA结合状态之间的构象变化发生在一个机械步骤，从初始形成或解离的DPO 4和DNA的二元复合物不同。
Numerous kinetic, structural, and theoretical studies have established that DNA polymerases adjust their domain structures to enclose nucleotides in their active sites and then rearrange critical active site residues and substrates for catalysis, with the latter conformational change acting to kinetically limit the correct nucleotide incorporation rate. Additionally, structural studies have revealed a large conformational change between the apoprotein and the DNA–protein binary state for Y-family DNA polymerases. In previous studies [Xu, C., Maxwell, B. A., Brown, J. A., Zhang, L., and Suo, Z. (2009) PLoS Biol.7, e1000225], a real-time Förster resonance energy transfer (FRET) method was developed to monitor the global conformational transitions of DNA polymerase IV from Sulfolobus solfataricus (Dpo4), a prototype Y-family enzyme, during nucleotide binding and incorporation by measuring changes in distance between locations on the enzyme and the DNA substrate. To elucidate further details of the conformational transitions of Dpo4 during substrate binding and catalysis, in this study, the real-time FRET technique was used to monitor changes in distance between various pairs of locations in the protein itself. In addition to providing new insight into the conformational changes as revealed in previous studies, the results here show that the previously described conformational change between the apo and DNA-bound states of Dpo4 occurs in a mechanistic step distinct from initial formation or dissociation of the binary complex of Dpo4 and DNA.
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