Mullins Elwood A, Salay Lauren E, Durie Clarissa L, Bradley Noah P, Jackman Jane E, Ohi Melanie D, Chazin Walter J, Eichman Brandt F
bioRxiv. 2023 Sep 28:2023.03.16.533013. doi: 10.1101/2023.03.16.533013.
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 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-引发酶（polα-引发酶）合成具有确定长度和组成的嵌合RNA-DNA引物的机制尚不清楚，而这种引物对于复制保真度和基因组稳定性是必需的。在此，我们报告了polα-引发酶与代表DNA合成不同阶段的引发模板复合物的冷冻电镜结构。我们的数据表明，引发酶调节亚基与引物5'-末端的相互作用如何促进引物向polα的交接并增加polα的持续合成能力，从而调节RNA和DNA的组成。这些结构详细说明了异源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DNA合成的终止是通过降低polα和引发酶对沿着嵌合引物/模板双链体的不同构象的亲和力来促进的。总之，这些发现阐明了复制起始中的一个关键催化步骤，并为polα-引发酶合成引物提供了一个全面的模型。
