Regulated complex assembly safeguards the fidelity of Sleeping Beauty transposition

The functional relevance of the inverted repeat structure (IR/DR) in a subgroup of the Tc1/mariner superfamily of transposons has been enigmatic. In contrast to mariner transposition, where a topological filter suppresses single-ended reactions, the IR/DR orchestrates a regulatory mechanism to enforce synapsis of the transposon ends before cleavage by the transposase occurs. This ordered assembly process shepherds primary transposase binding to the inner 12DRs (where cleavage does not occur), followed by capture of the 12DR of the other transposon end. This extra layer of regulation suppresses aberrant, potentially genotoxic recombination activities, and the mobilization of internally deleted copies in the IR/DR subgroup, including Sleeping Beauty (SB). In contrast, internally deleted sequences (MITEs) are preferred substrates of mariner transposition, and this process is associated with the emergence of Hsmar1-derived miRNA genes in the human genome. Translating IR/DR regulation to in vitro evolution yielded an SB transposon version with optimized substrate recognition (pT4). The ends of SB transposons excised by a K248A excision⁺/integration^- transposase variant are processed by hairpin resolution, representing a link between phylogenetically, and mechanistically different recombination reactions, such as V(D)J recombination and transposition. Such variants generated by random mutation might stabilize transposon-host interactions or prepare the transposon for a horizontal transfer.