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

    Keywords

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

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