利用磁镊对拓扑异构酶活性进行单分子测量。

Single-molecule measurements of topoisomerase activity with magnetic tweezers.

作者信息

Seol Yeonee, Neuman Keir C

机构信息

Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

出版信息

Methods Mol Biol. 2011;778:229-41. doi: 10.1007/978-1-61779-261-8_15.

DOI:10.1007/978-1-61779-261-8_15

PMID:21809210

Abstract

Magnetic tweezers provide a versatile tool enabling the precise application of force and torque on -individual biomolecules. These properties make magnetic tweezers uniquely suited for the study of DNA topology and topoisomerases at the single-molecule level. Single-molecule approaches, which are complementary to ensemble biochemical and structural approaches, have provided remarkable insights into the mechanisms of topoisomerase activity and interactions with DNA. Here, we describe how to make single-molecule measurements of topoisomerase activity with a magnetic tweezers instrument. We provide detailed instructions for preparing and characterizing DNA substrates, flow cells, and supercoilable DNA tethers. We then describe magnetic tweezers measurements of supercoil relaxation by single topoisomerases.

摘要

磁镊提供了一种多功能工具，能够对单个生物分子精确施加力和扭矩。这些特性使得磁镊特别适用于在单分子水平上研究DNA拓扑结构和拓扑异构酶。单分子方法与整体生化和结构方法相辅相成，为拓扑异构酶活性机制及其与DNA相互作用提供了显著的见解。在这里，我们描述了如何使用磁镊仪器对拓扑异构酶活性进行单分子测量。我们提供了制备和表征DNA底物、流动池和可超螺旋化DNA系链的详细说明。然后，我们描述了通过单个拓扑异构酶进行超螺旋松弛的磁镊测量。