First Biochemical Steps on Bacterial Transposition Pathways

Catherine Guynet, Emilien Nicolas, Bao Ton-Hoang, Jean Yves Bouet, Bernard Hallet

Research output: Contribution in Book/Catalog/Report/Conference proceedingChapter

Abstract

Transposons are found in a wide variety of forms throughout the prokaryotic world where they actively contribute to the adaptive strategies of bacterial communities and hence, to the continuous emergence of new multiresistant pathogens. Contrasting with their biological and societal impact, only a few bacterial transposons have been the subject of detailed molecular studies. In this chapter, we propose a set of reliable biochemical methods as a primary route for studying new transposition mechanisms. These methods include (a) a straightforward approach termed “thermal shift induction” to produce the transposase in a soluble and properly folded configuration prior to its purification, (b) an adaptation of classical electrophoretic mobility shift assays (EMSA) combined to fluorescently labeled DNA substrates to determine the DNA content of different complexes assembled by the transposase, and (c) a highly sensitive “in-gel” DNA footprinting assay to further characterize these complexes at the base pair resolution level. A combination of these approaches was recently applied to decipher the molecular organization of key intermediates in the Tn3-family transposition pathway, a mechanism that has long been refractory to biochemical studies.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages157-177
Number of pages21
DOIs
Publication statusPublished - 1 Jan 2020

Publication series

NameMethods in Molecular Biology
Volume2075
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Fingerprint

Transposases
DNA Footprinting
DNA
Electrophoretic Mobility Shift Assay
Base Pairing
Hot Temperature
Gels

Keywords

  • DNA binding
  • DNA–protein complex
  • EMSA
  • Fluorescent DNA labeling
  • In-gel DNA footprinting
  • Nucleoprotein complexes stoichiometry
  • Thermal-shift induction
  • Transposase purification
  • Transpososome

Cite this

Guynet, C., Nicolas, E., Ton-Hoang, B., Bouet, J. Y., & Hallet, B. (2020). First Biochemical Steps on Bacterial Transposition Pathways. In Methods in Molecular Biology (pp. 157-177). (Methods in Molecular Biology; Vol. 2075). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9877-7_12
Guynet, Catherine ; Nicolas, Emilien ; Ton-Hoang, Bao ; Bouet, Jean Yves ; Hallet, Bernard. / First Biochemical Steps on Bacterial Transposition Pathways. Methods in Molecular Biology. Humana Press Inc., 2020. pp. 157-177 (Methods in Molecular Biology).
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Guynet, C, Nicolas, E, Ton-Hoang, B, Bouet, JY & Hallet, B 2020, First Biochemical Steps on Bacterial Transposition Pathways. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 2075, Humana Press Inc., pp. 157-177. https://doi.org/10.1007/978-1-4939-9877-7_12

First Biochemical Steps on Bacterial Transposition Pathways. / Guynet, Catherine; Nicolas, Emilien; Ton-Hoang, Bao; Bouet, Jean Yves; Hallet, Bernard.

Methods in Molecular Biology. Humana Press Inc., 2020. p. 157-177 (Methods in Molecular Biology; Vol. 2075).

Research output: Contribution in Book/Catalog/Report/Conference proceedingChapter

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Guynet C, Nicolas E, Ton-Hoang B, Bouet JY, Hallet B. First Biochemical Steps on Bacterial Transposition Pathways. In Methods in Molecular Biology. Humana Press Inc. 2020. p. 157-177. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-9877-7_12