Cryo-EM structures of DNA Polymerase Zeta in Translesion DNA Synthesis

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 was unknown. We resolved structures of the Polζ holoenzyme by single particle cryo-EM but first had to overcome the challenge of preferred orientation. We succeeded in capturing Polζ in the act of DNA synthesis, as well as without DNA. 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 uncovered features that impose high fidelity during the nucleotide-incorporation step and those that accommodate DNA lesions and mismatches during the extension reaction. Collectively, the structures decrypt the molecular underpinnings of Polζ’s role in TLS and provide a framework for new cancer therapeutics.