Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy.
Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy.
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DOI：
10.1021/nl502890g
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发表时间：
2014-10-08
期刊：
影响因子：
10.8
通讯作者：
Spies M
中科院分区：
文献类型：
--
作者：
Ghoneim M;Spies M
我们报道了一种双重照明的单分子成像策略，可以直接和实时地剖析DNA修复蛋白的纳米级结构域运动与其与单个DNA底物相互作用之间的关系。该策略被应用于含有FeS簇的DNA修复解旋酶XPD。通过FeS介导的XPD特异结合的荧光团位点的猝灭来评估构象动力学。同时，通过DNA和蛋白质衍生信号的共定位，检测到标记有光谱不同荧光团的DNA分子的结合。我们发现XPD经历了热驱动的构象转变，这种转变表现在它的两个辅助域的空间分离。DNA结合并不严格强制特定的构象。然而，与同源DNA损伤的相互作用，通过使这种状态的加权平均寿命比未损伤的DNA增加140%，稳定了XPD的紧密构象。我们的成像策略将是研究其他含FeS的核酸处理酶的有价值的工具。
We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes.
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