Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, Madrid, Spain.
Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, Madrid, Spain.
Enzymes. 2021;49:115-148. doi: 10.1016/bs.enz.2021.09.001. Epub 2021 Oct 13.
Faithfull replication of genomic information relies on the coordinated activity of the multi-protein machinery known as the replisome. Several constituents of the replisome operate as molecular motors that couple thermal and chemical energy to a mechanical task. Over the last few decades, in vitro single-molecule manipulation techniques have been used to monitor and manipulate mechanically the activities of individual molecular motors involved in DNA replication with nanometer, millisecond, and picoNewton resolutions. These studies have uncovered the real-time kinetics of operation of these biological systems, the nature of their transient intermediates, and the processes by which they convert energy to work (mechano-chemistry), ultimately providing new insights into their inner workings of operation not accessible by ensemble assays. In this chapter, we describe two of the most widely used single-molecule manipulation techniques for the study of DNA replication, optical and magnetic tweezers, and their application in the study of the activities of proteins involved in viral DNA replication.
基因组信息的忠实复制依赖于多蛋白机器——复制体的协调活动。复制体的几个组成部分作为分子马达运作，将热能和化学能转化为机械任务。在过去的几十年中，体外单分子操纵技术已被用于以纳米、毫秒和皮牛顿分辨率监测和操纵参与 DNA 复制的单个分子马达的机械活动。这些研究揭示了这些生物系统实时运行的动力学、它们的瞬态中间产物的性质，以及它们将能量转化为功（机械化学）的过程，最终为理解其内在运作提供了新的见解，这些是通过整体分析无法获得的。在本章中，我们描述了两种最广泛用于研究 DNA 复制的单分子操纵技术，即光学和磁镊，以及它们在研究参与病毒 DNA 复制的蛋白质的活性方面的应用。
