Cheng Yingxian, Han Pu, Peng Qi, Liu Ruili, Liu Hao, Yuan Bin, Zhao Yunfei, Kuai Lu, Qi Jianxun, Miao Kai, Shi Yi, Gao George Fu, Wang Han
College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan, China.
Beijing Life Science Academy, Beijing, China.
Nat Commun. 2025 Jun 4;16(1):5176. doi: 10.1038/s41467-025-60539-1.
The replicative helicase-catalyzed unwinding of the DNA double helix is the initiation of DNA replication. Helicases and primases are functionally related enzymes that have even been expressed as fusion proteins in some organisms and viruses. However, the mechanism underlying DNA unwinding initiation by these helicase-primase fusion enzymes and the functional association between domains have not been elucidated. Herein, we report the cryo-EM structures of mpox virus E5, the founding member of these helicase-primase enzymes, in various enzymatic stages. Notably, E5 forms a head-to-head double hexamer encircling dsDNA, disrupted by the conformational rearrangement of primase domains upon nucleotide incorporation. Five E5-ssDNA-ATP structures further support an ATP cycle-driven non-classical escort model for E5 translocation. Finally, the helicase domain is found to enhance the primase function as a DNA scaffold. Together, our data shed light on the E5-mediated DNA unwinding model including dsDNA loading, DNA melting, ssDNA translocation, and provide a reasonable interpretation for evolutionary preservation of helicase-primase fusion from a functional perspective.
复制性解旋酶催化的DNA双螺旋解旋是DNA复制的起始过程。解旋酶和引发酶是功能相关的酶，在某些生物体和病毒中甚至被表达为融合蛋白。然而，这些解旋酶-引发酶融合酶启动DNA解旋的机制以及结构域之间的功能关联尚未阐明。在此，我们报道了这些解旋酶-引发酶的创始成员——猴痘病毒E5在不同酶促阶段的冷冻电镜结构。值得注意的是，E5形成一个头对头的双六聚体环绕双链DNA，在核苷酸掺入时引发酶结构域的构象重排会破坏这种结构。五个E5-单链DNA-ATP结构进一步支持了E5易位的ATP循环驱动的非经典护送模型。最后，发现解旋酶结构域作为DNA支架增强了引发酶的功能。总之，我们的数据揭示了E5介导的DNA解旋模型，包括双链DNA加载、DNA解链、单链DNA易位，并从功能角度为解旋酶-引发酶融合的进化保守性提供了合理的解释。
