Transposition with Tn3-family elements occurs through interaction with the host β-sliding clamp processivity factor.

Tn3 family transposons are a widespread group of replicative transposons, notorious for contributing to the dissemination of antibiotic resistance, particularly the global prevalence of carbapenem resistance. The transposase (TnpA) of these elements catalyzes DNA breakage and rejoining reactions required for transposition. However, the molecular mechanism for target site selection with these elements remains unclear. Here, we identify a QLxxLR motif in N-terminal of Tn3 TnpAs and demonstrate that this motif allows interaction between TnpA of Tn3 family transposon Tn1721 and the host β-sliding clamp (DnaN), the major processivity factor of the DNA replication machinery. The TnpA-DnaN interaction is essential for Tn1721 transposition. Our work unveils a mechanism whereby Tn3 family transposons can bias transposition into certain replisomes through an interaction with the host replication machinery. This study further expands the diversity of mobile elements that use interaction with the host replication machinery to bias integration.