During chromosome replication, the nascent leading strand is synthesized by DNA polymerase epsilon (Pol ε), which associates with the sliding clamp processivity factor proliferating cell nuclear antigen (PCNA) to form a processive holoenzyme. For high-fidelity DNA synthesis, Pol ε relies on nucleotide selectivity and its proofreading ability to detect and excise a misincorporated nucleotide. Here, we present cryo-electron microscopy (cryo-EM) structures of human Pol ε in complex with PCNA, DNA and an incoming nucleotide, revealing how Pol ε associates with PCNA through its PCNA-interacting peptide box and additional unique features of its catalytic domain. Furthermore, by solving a series of cryo-EM structures of Pol ε at a mismatch-containing DNA, we elucidate how Pol ε senses and edits a misincorporated nucleotide. Our structures delineate steps along an intramolecular switching mechanism between polymerase and exonuclease activities, providing the basis for a proofreading mechanism in B-family replicative polymerases.

在染色体复制过程中，新生的前导链由 DNA 聚合酶 ε （Pol ε） 合成，它与滑动钳持续合成因子增殖细胞核抗原 （PCNA） 结合形成进行过程中的全酶。对于高保真 DNA 合成，Pol ε 依赖于核苷酸选择性及其校对能力来检测和切除错误掺入的核苷酸。在这里，我们展示了人 Pol ε 与 PCNA、DNA 和传入核苷酸复合的冷冻电子显微镜 （cryo-EM） 结构，揭示了 Pol ε 如何通过其 PCNA 相互作用的肽盒及其催化结构域的其他独特特征与 PCNA 结合。此外，通过解析 Pol ε 在包含错配的 DNA 上的一系列冷冻电镜结构，我们阐明了 Pol ε 如何感知和编辑错误掺入的核苷酸。我们的结构描绘了聚合酶和核酸外切酶活性之间分子内转换机制的步骤，为 B 家族复制聚合酶中的校对机制提供了基础。