First Biochemical Steps on Bacterial Transposition Pathways

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

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    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.

    langue originaleAnglais
    titreMethods in Molecular Biology
    EditeurHumana Press Inc.
    Nombre de pages21
    Les DOIs
    Etat de la publicationPublié - 1 janv. 2020

    Série de publications

    NomMethods in Molecular Biology
    ISSN (imprimé)1064-3745
    ISSN (Electronique)1940-6029

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