The proofreading mechanism of the human leading- strand DNA polymerase ε holoenzyme

• 成果类型: Article
• 署名作者: Wang, Feng; He, Qing; O'Donnell, Michael E.; Li, Huilin
• 署名单位: Van Andel Institute; Rockefeller University; Rockefeller University; Howard Hughes Medical Institute
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13127
• DOI: 10.1073/pnas.2507232122
• 发表日期: 2025-06-03
• 关键词: beta-hairpin loop; structural basis; cmg helicase; replication; FIDELITY; mutations; complexes; DYNAMICS; errors; diversity

摘要

The eukaryotic leading-strand DNA polymerase epsilon(Pol epsilon) is a dual-function enzyme with a proofreading 3 '-5 ' exonuclease (exo) site located 40 & Aring; from the DNA synthesizing pol site. Errors in Pol epsilon proofreading can cause various mutations, including C-to-G trans-versions, the most prevalent mutation in cancers and genetic diseases. Pol epsilon interacts with all three subunits of the PCNA ring to assemble a functional holoenzyme. Despite previous studies on proofreading of several Pol's, how Pol epsilon-or any Pol complexed with its sliding clamp-proofreads a mismatch generated in situ has been unknown. We show here by cryo-EM that a template/primer DNA substrate with a preexisting mismatch cannot enter the exo site of Pol epsilon-PCNA holoenzyme, but a mismatch generated in situ in the pol site yields three bona fide proofreading intermediates of Pol epsilon-PCNA holoenzyme. These intermediates reveal how the mismatch is dislodged from the pol site, how the DNA unwinds six base pairs, and how the unpaired primer 3 '-end is inserted into the exo site for cleavage. These results unexpectedly demonstrate that PCNA imposes strong steric constraints that extend unwinding and direct the trajectory of mismatched DNA and that this trajectory is dramatically different than for Pol epsilon in the absence of PCNA. These findings suggest a physiologically relevant proofreading mechanism for the human Pol epsilon holoenzyme.