Probing DNA transposition complex formation by AFM-based force spectroscopy

FERNANDEZ, MARICRUZ; ALSTEENS, DAVID; HALLET, BERNARD

Simposio; Approaches and Concepts in Microbiology, EMBO-EMBL Symposia; 2021

EMBO-EMBL

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which is remarkably prevalent in the dissemination of antibiotic resistances among pathogens. In spite of this, the molecular mechanisms that control the mobility of these elements are still poorly understood. Here, we use force−distance curve-based atomic force microscopy (FD-based AFM) to probe the binding of the transposase (TnpA) to DNA molecules containing one or two transposon terminal inverted repeats (IR) and to monitor transposition complex assembly dynamically at single-particle level. Analysis revealed that most interactions were mono-molecular, that is, between one TnpA molecule and one IR end to form a single end complex (SEC) with an average force of ~40 pN and a high affinity (508 nM). In addition, the presence of a sub-population of interactions was observed, corresponding to two SEC formation in parallel, therefore doubling the interaction force (~70 pN). Subsequently, a blocking DNA mimicking the second end of the transposon was used to study the binding inhibition properties. Addition of the molecule led to a significant binding reduction, providing direct evidence for SEC formation specificity. Interestingly, in experiments performed with DNA substrates containing two IR ends as in the native transposon, a third population was observed with an interaction force of ~120 pN. Here, a single TnpA molecule is thought join two IR ends of the substrate, forming a paired end complex (PEC). PEC formation followed first-order association kinetics with a time constant (τ) of ∼156 ms for TnpAWT. By contrast, PEC formation occurred faster in the case of hyperactive TnpA mutants that were previously shown to bind two transposon ends cooperatively. The results provide an unprecedented insight into how the transposition complex is assembled and becomes activated in order to catalyse transposition.