AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements

FERNANDEZ, MARICRUZ; SHKUMATOV, ALEXANDER V; LIU, YUN; STULEMEIJER, CLAIRE; DERCLAYE, SYLVIE; EFREMOV, ROUSLAN G; HALLET, BERNARD; ALSTEENS, DAVID

NUCLEIC ACIDS RESEARCH

OXFORD UNIV PRESS

Año: 2023

0305-1048

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, whichplays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recentdata on the structural architecture of the transposition complex, the molecular mechanisms underlyingthe replicative transposition of these elements arestill poorly understood. Here, we use force-distancecurve-based atomic force microscopy to probe thebinding of the TnpA transposase of Tn4430 to DNAmolecules containing one or two transposon endsand to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathwayfor transposition complex formation and activationduring which TnpA first binds as a dimer to a single transposon end and then undergoes a structuraltransition that enables it to bind the second end cooperatively and to become activated for transpositioncatalysis, the latter step occurring at a much fasterrate for the TnpA mutants. Our study thus providesan unprecedented approach to probe the dynamic ofa complex DNA processing machinery at the singleparticle level.