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-Structure paper
Title | AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements. |
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Journal, issue, pages | Nucleic Acids Res, Vol. 51, Issue 10, Page 4929-4941, Year 2023 |
Publish date | Jun 9, 2023 |
Authors | Maricruz Fernandez / Alexander V Shkumatov / Yun Liu / Claire Stulemeijer / Sylvie Derclaye / Rouslan G Efremov / Bernard Hallet / David Alsteens / |
PubMed Abstract | Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent ...Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent data on the structural architecture of the transposition complex, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds as a dimer to a single transposon end and then undergoes a structural transition that enables it to bind the second end cooperatively and to become activated for transposition catalysis, the latter step occurring at a much faster rate for the TnpA mutants. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level. |
External links | Nucleic Acids Res / PubMed:37026471 / PubMed Central |
Methods | EM (single particle) |
Resolution | 4.4 - 6.0 Å |
Structure data | EMDB-15213: Cryo-EM density map of Tn4430 TnpA hyperactive mutant (TnpA3X) in complex with IR48 substrate. EMDB-15218: Medium resolution cryo-EM density map of Tn4430 TnpA transposase from Tn3 family in apo state |
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