Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase

Tyler L. Dangerfield, Serdal Kirmizialtin, Kenneth A. Johnson

Research output: Contribution to journalArticlepeer-review


Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 30-50 proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioatemodified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.

Original languageEnglish (US)
Article number101627
JournalJournal of Biological Chemistry
Issue number3
StatePublished - Mar 1 2022


  • DNA/chemistry
  • DNA Replication
  • DNA-Directed DNA Polymerase/chemistry
  • Exonucleases
  • Structure-Activity Relationship
  • Substrate Specificity

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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