DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase

Nucleic Acids Res. 2007;35(16):5452-63. doi: 10.1093/nar/gkm591. Epub 2007 Aug 15.


DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s(-1); (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Aminopurine / chemistry
  • Amino Acid Substitution
  • Base Pair Mismatch
  • Binding Sites
  • Catalysis
  • DNA / biosynthesis
  • DNA / chemistry
  • DNA Replication*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Exodeoxyribonucleases / metabolism*
  • Fluorescence
  • Kinetics
  • Nucleotides / metabolism
  • Thymine / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*


  • Nucleotides
  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • 2-Aminopurine
  • DNA
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Thymine