Department of Biosciences, Rice University, Houston, TX, 77005, USA.
Nat Commun. 2022 Apr 29;13(1):2346. doi: 10.1038/s41467-022-30005-3.
Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis.
DNA 的无差错复制对生命至关重要。尽管几种聚合酶具有校对能力，但聚合酶的固有保真度通常远高于碱基配对能量所能提供的保真度。尽管研究人员已经通过动力学、结构测定和计算模拟研究了这个长期存在的问题，但决定聚合酶保真度的结构因素尚未完全解决。时分辨晶学阐明了正确的核苷酸掺入，并为聚合酶建立了一个三金属离子依赖的催化机制。使用 X 射线时分辨晶学，我们可视化了由 DNA 聚合酶 η 催化的完整 DNA 错误掺入过程。得到的分子快照表明，由 A 位金属离子结合介导的引物 3´-OH 排列是底物识别的关键步骤。此外，我们观察到 C 位金属离子结合先于核苷酸转移反应，并证明 C 位金属离子严格需要错误掺入。我们的结果强调了三个金属离子在 DNA 合成中的重要但独立的作用。
