Yuan Zuanning, Georgescu Roxana, Li Huilin, O'Donnell Michael E
Department of Structural Biology, Van Andel Institute, Grand Rapids, Michigan, USA.
Howard Hughes Medical Institute.
bioRxiv. 2023 May 3:2023.05.03.539257. doi: 10.1101/2023.05.03.539257.
The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.
真核生物聚合酶α（Polα）是一种具有双重功能的DNA聚合酶/引发酶复合物，它能合成一段20 - 30个核苷酸的RNA - DNA杂交引物用于DNA复制。Polα由Pol1、Pol12、引发酶1（Pri1）和Pri2组成，其中Pol1和Pri1分别具有DNA聚合酶活性和RNA引发酶活性，而Pol12和Pri2起结构作用。目前尚不清楚Polα如何将Pri1合成的RNA引物传递给Pol1以进行DNA引物延伸，以及引物长度是如何确定的，这可能是由于研究高度动态的结构存在困难。在此，我们报告了对完整的四亚基酵母Polα在无底物、引物起始、引物延伸、RNA引物从Pri1传递给Pol1以及DNA延伸状态下的全面冷冻电镜分析，分辨率范围为3.5 Å - 5.6 Å。我们发现Polα是一种三结构域的柔性结构。Pri2作为一个柔性铰链，将催化性的Pol1核心与非催化性的Pol1 C末端结构域连接在一起，后者与Pol12结合形成一个稳定的平台，其他组分在此平台上有序排列。在无底物状态下，Pol1核心被隔离在Pol12 - Pol1 - C末端结构域平台上，Pri1可能处于移动状态以寻找模板。一旦结合单链DNA模板，就会引发大的构象变化，使Pri1能够进行RNA合成，并将Pol1核心定位到Pri1结合位点上游50 Å处接受未来的RNA引发位点。我们详细揭示了Pol1核心从Pri1接管RNA 3'末端的关键点。DNA引物延伸似乎受到Pol1核心螺旋运动的限制，而Pri2 - C末端结构域则稳定地保持在RNA引物的5'末端。由于Pri1和Pol1核心都通过两个连接子连接到平台上，引物生长将在这种“两点”连接中产生应力，这可能会限制RNA - DNA杂交引物的长度。因此，这项研究揭示了Polα为合成DNA复制引物所经历的一系列大规模动态运动。
