The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently...

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.