The mechanism by which polymerase α–primase (polα–primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα–primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5′ end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer–template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα–primase.

聚合酶 α-primase (polα-primase) 合成具有确定的长度和组成的嵌合 RNA-DNA 引物的机制尚不清楚，这对于复制保真度和基因组稳定性是必需的。在这里，我们报道了非洲爪蟾polα-引物酶与代表 DNA 合成各个阶段的引物模板复合物的冷冻电镜结构。我们的数据显示引物酶调节亚基与引物 5' 末端的相互作用如何促进引物向 polα 的切换并增加 polα 的持续合成能力，从而调节 RNA 和 DNA 组成。这些结构详细说明了异四聚体内的灵活性如何实现跨两个活性位点的合成，并提供证据表明，通过减少 polα 和引物酶对嵌合引物-模板双链体上不同构象的亲和力，可以促进 DNA 合成的终止。总之，这些发现阐明了复制起始中的关键催化步骤，并为 polα-引物酶合成引物提供了综合模型。