DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31‚ Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein–protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ’s role in TLS and provide a framework for new cancer therapeutics.

DNA 聚合酶 ζ (Polζ) 与高保真复制聚合酶属于同一 B 家族，但专门用于转位 DNA 合成 (TLS) 中的延伸反应。尽管它在 TLS 中很重要，但 Polζ 的结构是未知的。我们展示了酿酒 酵母的冷冻电镜结构Polζ 全酶在 DNA 合成 (3.1 Å) 和没有 DNA (4.1 Å) 的情况下。Polζ 显示出五聚体环状结构，具有催化作用的 Rev3、附件 Pol31、Pol32 和两个 Rev7 亚基，形成不间断的蛋白质-蛋白质相互作用菊花链。我们还揭示了在核苷酸掺入步骤中强加高保真度的特征，以及在延伸反应中适应错配和损伤的特征。总的来说，我们解密了 Polζ 在 TLS 中的作用的分子基础，并为新的癌症治疗提供了一个框架。