Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole normale supérieure, CNRS, INSERM, PSL Research University, Paris, France.
Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole normale supérieure, CNRS, INSERM, PSL Research University, Paris, France; Laboratoire de Physique de L'Ecole Normale Supérieure de Paris, CNRS, ENS, Université PSL, Sorbonne Université, Université de Paris, Paris, France.
Methods Enzymol. 2022;673:359-403. doi: 10.1016/bs.mie.2022.03.035. Epub 2022 Apr 22.
Helicases form a universal family of molecular motors that bind and translocate onto nucleic acids. They are involved in essentially every aspect of nucleic acid metabolism: from DNA replication to RNA decay, and thus ensure a large spectrum of functions in the cell, making their study essential. The development of micromanipulation techniques such as magnetic tweezers for the mechanistic study of these enzymes has provided new insights into their behavior and their regulation that were previously unrevealed by bulk assays. These experiments allowed very precise measures of their translocation speed, processivity and polarity. Here, we detail our newest technological advances in magnetic tweezers protocols for high-quality measurements and we describe the new procedures we developed to get a more profound understanding of helicase dynamics, such as their translocation in a force independent manner, their nucleic acid binding kinetics and their interaction with roadblocks.
解旋酶形成了一个普遍的分子马达家族，能够结合并在核酸上移位。它们参与了核酸代谢的各个方面：从 DNA 复制到 RNA 降解，因此确保了细胞中大量的功能，使它们的研究至关重要。诸如磁镊之类的微操作技术的发展，为这些酶的机制研究提供了新的见解，这些见解是以前的批量分析所无法揭示的。这些实验非常精确地测量了它们的迁移速度、连续性和极性。在这里，我们详细介绍了我们在磁镊技术方面的最新技术进展，以实现高质量的测量，并描述了我们开发的新程序，以更深入地了解解旋酶的动力学，例如它们以不受力的方式进行迁移、核酸结合动力学以及与障碍物的相互作用。
