基于磁镊的单分子分析技术研究大肠杆菌 SSB 与 DNA 及 RecQ 解旋酶的相互作用。

Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and RecQ Helicase.

机构信息

Physics Department, Faculty of Science, Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India.

Laboratoire de Physique Statistique, Département de physique de l'ENS, École normale supérieure, PSL Research University, Université Paris Diderot, Sorbonne Paris Cité, Sorbonne Universités, UPMC University Paris 06, CNRS, Paris, France.

出版信息

Methods Mol Biol. 2021;2281:93-115. doi: 10.1007/978-1-0716-1290-3_6.

DOI:10.1007/978-1-0716-1290-3_6

PMID:33847954

Abstract

The ability of magnetic tweezers to apply forces and measure molecular displacements has resulted in its extensive use to study the activity of enzymes involved in various aspects of nucleic acid metabolism. These studies have led to the discovery of key aspects of protein-protein and protein-nucleic acid interaction, uncovering dynamic heterogeneities that are lost to ensemble averaging in bulk experiments. The versatility of magnetic tweezers lies in the possibility and ease of tracking multiple parallel single-molecule events to yield statistically relevant single-molecule data. Moreover, they allow tracking both fast millisecond dynamics and slow processes (spanning several hours). In this chapter, we present the protocols used to study the interaction between E. coli SSB, single-stranded DNA (ssDNA), and E. coli RecQ helicase using magnetic tweezers. In particular, we propose constant force and force modulation assays to investigate SSB binding to DNA, as well as to characterize various facets of RecQ helicase activity stimulation by SSB.

摘要

磁镊能够施加力并测量分子位移，这使其广泛应用于研究参与核酸代谢各个方面的酶的活性。这些研究导致了发现蛋白质-蛋白质和蛋白质-核酸相互作用的关键方面，揭示了在批量实验中由于整体平均而丢失的动态异质性。磁镊的多功能性在于跟踪多个平行单分子事件的可能性和易用性，从而产生具有统计学意义的单分子数据。此外，它们允许跟踪快速毫秒级动力学和缓慢过程（跨越数小时）。在本章中，我们介绍了使用磁镊研究大肠杆菌 SSB、单链 DNA (ssDNA) 和大肠杆菌 RecQ 解旋酶之间相互作用的实验方案。特别是，我们提出了恒力和力调制测定法来研究 SSB 与 DNA 的结合，以及表征 SSB 对 RecQ 解旋酶活性的各种方面的刺激。