Molecular Dynamics Reveal Base Flipping as a Key Mechanism in Tc3a Transposase DNA Recognition

Transposons, or "jumping genes," are mobile genetic elements that reshape genomes. The Tc1/Mariner family, including Tc3a transposase, has been studied for decades, but key aspects of its DNA recognition and cleavage remain unresolved. Existing models propose a homo-dimeric process for DNA excision and integration, yet conflicting hypotheses exist about the initial steps of DNA cleavage and target site recognition. Here, we reveal a previously unrecognized base-flipping mechanism in Tc3a transposase that challenges long-standing models of Mariner transposon activity. Using molecular dynamics simulations, we demonstrate that N-terminal acetylation induces a structural shift in the transposase-DNA complex, forcing a nucleotide in the inverted repeat to flip outward. This base-flipping event alters the local DNA conformation, creating torsional strain that may facilitate transposon inverted terminal repeat association. Unlike prior models, which assumed the entire protein, or protein dimers, were needed to illicit DNA interactions necessary for any types of torsion, our findings indicate that transposase bipartite binding itself actively reshapes DNA structure. We further show that this mechanism is dependent on the bipartite helix-turn-helix domains, which differentially contribute to DNA stabilization and recognition. Our simulations also reveal that the processed N-terminus plays an unexpected role in modulating DNA binding affinity, contradicting previous assumptions that it was structurally inconsequential. Together, the two helix-turn-helix motifs act to propagate the force caused by base flipping along one direction of the cognate DNA double helix axis. The fact that previous structural studies lacked this amino acid modification may explain why this mechanism was overlooked. These results provide a new framework for understanding transposase-DNA interactions, highlighting how a single molecular modification can trigger major DNA rearrangements. This discovery not only redefines the Tc3a transposition process but also calls for a reassessment of long-standing Mariner family transposon models.