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.

Helitrons是广泛存在的真核 DNA 转座子，对基因组变异和进化做出了重大贡献，部分原因在于它们独特的复制滚环机制，这种机制经常动员相邻的基因。尽管大多数真核转座酶形成寡聚体并使用 RNase H 样结构域来破坏和重新连接双链 DNA (dsDNA)，但 Helitron转座酶包含单链 DNA (ssDNA) 特异性 HUH 核酸内切酶结构域。在这里，我们报告了Helitron的低温电子显微镜结构转座酶与 5'-转座子末端结合，提供对其多域结构和功能的深入了解。单体转座酶形成一个紧密堆积的组装体，将共价连接的切割末端掩埋，保护它直到第二个末端可用。该结构揭示了与 TraI 出乎意料的结构相似性，TraI 是一种也催化 ssD​​NA 运动的细菌松弛酶。HUH 活性位点表明两个并列的酪氨酸（许多使用 HUH 核酸酶的复制引发剂的一个特征）如何耦合 α-螺旋的构象转变以控制链切割和连接反应。