Abstract
Copy‐out/paste‐in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy‐out/paste‐in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X‐ray structures of ISC th4 transposase, a member of the IS 256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N‐terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA‐binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.
Highlights
Transposons are mobile genetic elements found in all living organisms and are important evolutionary shaping forces (Biemont & Vieira, 2006)
Only a handful of copy-out/paste-in TEs have been studied in detail and there is no mechanistic information on any element from this group that might provide insight into their unique strand transfer reactions
The second is the characterization of a four-a-helix insertion domain whose coordinated movement with the RNase H-like catalytic domain likely controls the progression of the transposition reaction
Summary
Transposons (or transposable elements, TE) are mobile genetic elements found in all living organisms and are important evolutionary shaping forces (Biemont & Vieira, 2006). Many ISs contain sequences that can act as promoters for genes located outside of the element and can dynamically affect their expression upon integration (Nevers & Saedler, 1977; reviewed in Siguier et al, 2015; Vandecraen et al, 2017; Babakhani & Oloomi, 2018). These properties of ISs have contributed to the rise of multidrug-resistant bacterial strains defined as urgent threats by the Centers for Disease Control and Prevention (Alekshun & Levy, 2007; McKenna, 2013; Watkins & Bonomo, 2016; CDC 2019) such as antibiotic-resistant C. difficile, and carbapenem-resistant Enterobacteriaceae and N. gonorrhoeae. Such understanding may provide an opportunity to develop novel gene delivery and modification tools for use in research as well as human medicine (Haapa et al, 1999; Izsvak & Ivics, 2004; Adey et al, 2010; Sakanaka et al, 2018)
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