44: Polε Proofreading Revealed

Wang F et al., PNAS - This episode examines a cryo-EM study that captures authentic proofreading intermediates of human Polε in complex with PCNA by generating a mismatch in situ. The work reveals how PCNA constrains DNA movement and how a mismatched primer is transferred from the polymerase to the exonuclease site. Key terms: DNA proofreading, DNA polymerase ε, PCNA, cryo-EM, replication fidelity. Study Highlights:Using cryo-EM of Polε–PCNA complexes with a mismatch made in situ, the authors captured three distinct proofreading intermediates (mismatch-locking, Pol-backtracking, and mismatch-editing). A preexisting mismatched T/P could not access the exo site when PCNA was engaged, showing PCNA imposes steric constraints on DNA movement. Proofreading proceeds with unwinding of six base pairs, template scrunching that forms out-of-register base pairs, and sustained Polε–PCNA association during transfer of the 3′ primer end to the exo site. Conclusion:The study defines a PCNA-constrained, processive proofreading pathway for human Polε involving 6-bp unwinding and out-of-register base pairing; disruptions of Polε–PCNA interactions or exonuclease function could alter replication fidelity and contribute to mutagenesis. Music:Enjoy the music based on this article at the end of the episode. Article title:The proofreading mechanism of the human leading-strand DNA polymerase ε holoenzyme First author:Wang F Journal:PNAS DOI:10.1073/pnas.2507232122 Reference:Wang F., He Q., O'Donnell M.E., Li H. The proofreading mechanism of the human leading-strand DNA polymerase ε holoenzyme. PNAS. 2025;122:e2507232122. doi:10.1073/pnas.2507232122 License:This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/ Support:Base by Base – Stripe donations: https://donate.stripe.com/7sY4gz71B2sN3RWac5gEg00 Official website https://basebybase.com On PaperCast Base by Base you'll discover the latest in genomics, functional genomics, structural genomics, and proteomics. Episode link: https://basebybase.com/episodes/pol-epsilon-proofreading-pnas-2025 QC:This episode was checked against the original article PDF and publication metadata for the episode release published on 2025-06-12. QC Scope:- article metadata and core scientific claims from the narration- excludes analogies, intro/outro, and music- transcript coverage: Audited the transcript sections describing the Polε–PCNA proofreading mechanism, in situ mismatch strategy, the three intermediates (mismatch-locking, backtracking, editing), the 6 bp unwinding/scrunching, structural roles (P-domain, thumb, linker), Pro286 wedge, and cancer-relevant mutations; plus implications and fut- transcript topics: Polε–PCNA proofreading mechanism; In situ mismatch generation and cryo-EM capture; Three proofreading intermediates; 6 bp unwinding and DNA scrunching; Role of P-domain, thumb, and linker helix; Pro286 wedge and cancer mutations QC Summary:- factual score: 10/10- metadata score: 10/10- supported core claims: 7- claims flagged for review: 0- metadata checks passed: 4- metadata issues found: 0 Metadata Audited:- article_doi- article_title- article_journal- license Factual Items Audited:- Polε–PCNA holoenzyme proofreading mechanism with in situ mismatch- Preexisting mismatches blocked from entering exo site when PCNA is engaged- Three proofreading intermediates observed: mismatch-locking, Pol-backtracking, mismatch-editing- 6 bp unwinding of template/primer during proofreading- Primer 3′-end translocates ~15 Å to reach the exo site- P-domain and linker-helix movements...