1. Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and RecQ Helicase
- Author
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Vincent Croquette, Debjani Bagchi, Bertrand Ducos, Maria Manosas, Samar Hodeib, and Weiting Zhang
- Subjects
0303 health sciences ,Magnetic tweezers ,RecQ helicase ,Ensemble averaging ,Nucleic acid metabolism ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,Biophysics ,Molecule ,Constant force ,030217 neurology & neurosurgery ,DNA ,030304 developmental biology - 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.
- Published
- 2021
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