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

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Single Stranded DNA Binding Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2281))

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.

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Authors and Affiliations

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

    Debjani Bagchi

  2. 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

    Weiting Zhang, Bertrand Ducos & Vincent Croquette

  3. IBENS, Département de biologie, École normale supérieure, CNRS, INSERM, PSL Research University, Paris, France

    Weiting Zhang, Bertrand Ducos & Vincent Croquette

  4. High Throughput qPCR Core Facility of the ENS, qPCR-HD-GPC, Université PSL, CNRS, IBENS, 46 rue d’Ulm, Paris, France

    Samar Hodeib & Bertrand Ducos

  5. Small Biosystems Lab, Departament de Física de la Matèria Condensada, Facultat de Física, Universitat de Barcelona, Barcelona, Spain

    Maria Manosas

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  1. Debjani Bagchi
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  2. Weiting Zhang
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  3. Samar Hodeib
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  4. Bertrand Ducos
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  5. Vincent Croquette
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  6. Maria Manosas
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Editors and Affiliations

  1. Jaboticabal, Brazil

    Marcos T. Oliveira

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Bagchi, D., Zhang, W., Hodeib, S., Ducos, B., Croquette, V., Manosas, M. (2021). Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and RecQ Helicase. In: Oliveira, M.T. (eds) Single Stranded DNA Binding Proteins. Methods in Molecular Biology, vol 2281. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1290-3_6

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  • DOI: https://doi.org/10.1007/978-1-0716-1290-3_6

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  • Print ISBN: 978-1-0716-1289-7

  • Online ISBN: 978-1-0716-1290-3

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