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Article Dans Une Revue Proceedings of the National Academy of Sciences of the United States of America Année : 2022

A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches

Résumé

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA–protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme—Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)—that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization ( Rut ) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating in vitro and in vivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.
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Dates et versions

hal-03800009 , version 1 (06-10-2022)

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Mildred Delaleau, Eric Eveno, Isabelle Simon, Annie Schwartz, Marc Boudvillain. A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119 (38), ⟨10.1073/pnas.2209608119⟩. ⟨hal-03800009⟩
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