Sun B, Wang M D
School of Life Science and Technology, ShanghaiTech University, Shanghai, PR China.
Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, NY, United States; Howard Hughes Medical Institute, Cornell University, Ithaca, NY, United States.
Methods Enzymol. 2017;582:55-84. doi: 10.1016/bs.mie.2016.08.001. Epub 2016 Oct 27.
The replisome is a multiprotein molecular machinery responsible for the replication of DNA. It is composed of several specialized proteins each with dedicated enzymatic activities, and in particular, helicase unwinds double-stranded DNA and DNA polymerase catalyzes the synthesis of DNA. Understanding how a replisome functions in the process of DNA replication requires methods to dissect the mechanisms of individual proteins and of multiproteins acting in concert. Single-molecule optical-trapping techniques have proved to be a powerful approach, offering the unique ability to observe and manipulate biomolecules at the single-molecule level and providing insights into the mechanisms of molecular motors and their interactions and coordination in a complex. Here, we describe a practical guide to applying these techniques to study the dynamics of individual proteins in the bacteriophage T7 replisome, as well as the coordination among them. We also summarize major findings from these studies, including nucleotide-specific helicase slippage and new lesion bypass pathway in T7 replication.
复制体是一种负责DNA复制的多蛋白分子机器。它由几种具有专门酶活性的特殊蛋白质组成，特别是解旋酶解开双链DNA，DNA聚合酶催化DNA的合成。了解复制体在DNA复制过程中的功能需要剖析单个蛋白质以及协同作用的多蛋白机制的方法。单分子光镊技术已被证明是一种强大的方法，它具有在单分子水平观察和操纵生物分子的独特能力，并能深入了解分子马达的机制及其在复合物中的相互作用和协调。在这里，我们描述了将这些技术应用于研究噬菌体T7复制体中单个蛋白质的动力学及其之间协调作用的实用指南。我们还总结了这些研究的主要发现，包括T7复制中核苷酸特异性解旋酶滑移和新的损伤绕过途径。
