Novel approaches to study helicases using magnetic tweezers

Nadia Ruiz-Gutierrez (1) , Martin Rieu (1) , Jimmy Ouellet (2) , Jean-François Allemand (1, 3) , Vincent Croquette (1, 4, 5) , Hervé Le Hir (1)

1 IBENS - Institut de biologie de l'ENS Paris
2 Depixus SAS [Paris]
3 Physique Multi-échelles du vivant
4 ABCD : Biophysique des Biomolécules
5 ESPCI Paris - Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris

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.

Mots clés: Magnetic tweezers; Helicases; DNA/RNA hairpins; Click chemistry surfaces; Three-way junction; Recombinant protein purification; Single molecule binding assay; Kinetic locking; Roadblock interaction

Domaines: Physique [physics]; Sciences du Vivant [q-bio]

Nadia Ruiz-Gutierrez, Martin Rieu, Jimmy Ouellet, Jean-François Allemand, Vincent Croquette, et al.. Novel approaches to study helicases using magnetic tweezers. Helicase Enzymes Part B, Elsevier, pp.359-403, 2022, Methods in Enzymology, ⟨10.1016/bs.mie.2022.03.035⟩. ⟨hal-03727451⟩