Single-molecule sorting of DNA helicases
Single-molecule sorting of DNA helicases
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DOI：
10.1016/j.ymeth.2016.05.009
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发表时间：
2016-10-01
期刊：
影响因子：
4.8
通讯作者：
Spies, Maria
中科院分区：
文献类型：
--
作者：
Bain, Fletcher E.;Wu, Colin G.;Spies, Maria
DNA解旋酶参与细胞DNA代谢的几乎所有方面，通过使用ATP-推动的沿DNA分子的定向移位沿着来解链DNA双链体、拆除核蛋白复合物和去除非规范DNA结构。翻译后修饰和解旋酶相互作用配偶体通常被视为控制真正的解旋酶活性和不涉及双链体分离的酶的其他功能之间的转换的决定因素。发展人类解旋酶及其翻译后修饰控制机制的瓶颈是获得足够数量的经修饰的解旋酶用于传统的结构功能分析和生化重建。这种限制可以通过单分子分析来克服，其中几百个表面束缚的分子足以获得解旋酶介导的底物结合和重排的完整动力学和热力学描述。用Cy 3和Cy 5荧光团位点特异性标记的合成寡核苷酸可用于产生多种DNA底物，其可用于表征DNA结合以及解旋酶易位和双链体解旋活性。本章介绍了“单分子分选”，一个强大的实验方法，同时量化，并区分其天然翻译后修饰的解旋酶的活动。使用这种技术，可以产生感兴趣的DNA解旋酶，并在人类细胞中进行生物素化，从而通过全内反射荧光显微镜进行单分子研究的表面束缚。从细胞中提取的解旋酶池预计含有后修饰和未修饰酶的混合物，并且可以分别监测来自任一群体的贡献，但在同一实验中提供了评估给定修饰效果的直接途径。(C)2016 Elsevier Inc. All rights reserved.
DNA helicases participate in virtually all aspects of cellular DNA metabolism by using ATP-fueled directional translocation along the DNA molecule to unwind DNA duplexes, dismantle nucleoprotein complexes, and remove non-canonical DNA structures. Post-translational modifications and helicase interacting partners are often viewed as determining factors in controlling the switch between bona fide helicase activity and other functions of the enzyme that do not involve duplex separation. The bottleneck in developing a mechanistic understanding of human helicases and their control by post-translational modifications is obtaining sufficient quantities of the modified helicase for traditional structure functional analyses and biochemical reconstitutions. This limitation can be overcome by single molecule analysis, where several hundred surface-tethered molecules are sufficient to obtain a complete kinetic and thermodynamic description of the helicase-mediated substrate binding and rearrangement. Synthetic oligonucleotides site-specifically labeled with Cy3 and Cy5 fluorophores can be used to create a variety of DNA substrates that can be used to characterize DNA binding, as well as helicase translocation and duplex unwinding activities. This chapter describes "single-molecule sorting", a robust experimental approach to simultaneously quantify, and distinguish the activities of helicases carrying their native post translational modifications. Using this technique, a DNA helicase of interest can be produced and biotinylated in human cells to enable surface-tethering for the single-molecule studies by total internal reflection fluorescence microscopy. The pool of helicases extracted from the cells is expected to contain a mixture of post-translationally modified and unmodified enzymes, and the contributions from either population can be monitored separately, but in the same experiment providing a direct route to evaluating the effect of a given modification. (C) 2016 Elsevier Inc. All rights reserved.