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Related Experiment Videos

Processive proofreading is intrinsic to T4 DNA polymerase.

M K Reddy1, S E Weitzel, P H von Hippel

  • 1Institute of Molecular Biology, University of Oregon, Eugene 97403.

The Journal of Biological Chemistry
|July 15, 1992
PubMed
Summary

Bacteriophage T4 DNA polymerase can efficiently proofread and continue DNA synthesis after removing mismatched bases. This editing process is highly processive for one or two errors, revealing insights into polymerase function.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA replication requires high processivity for accurate genome duplication.
  • Isolated DNA polymerases often exhibit lower processivity than in vivo complexes.
  • Bacteriophage T4 DNA polymerase possesses a proofreading exonuclease activity.

Purpose of the Study:

  • To investigate the processivity of T4 DNA polymerase during proofreading.
  • To determine if T4 DNA polymerase can switch between exonuclease and polymerase activity without dissociation.
  • To understand the mechanism of mismatched nucleotide removal and subsequent synthesis.

Main Methods:

  • Utilized mismatched primer/template substrates to mimic misincorporation events.
  • Employed single-turnover conditions with a heparin trap.

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  • Assessed the polymerase's ability to edit and resume synthesis.

    • Main Results:

    • T4 DNA polymerase removes misincorporated bases with >80% probability and continues synthesis processively.
    • Processive synthesis follows the removal of one or two mismatched nucleotides.
    • Processivity is significantly reduced when removing a triply mispaired terminus.

    Conclusions:

    • The editing active site of T4 DNA polymerase optimally accommodates two unpaired nucleotides.
    • Editing efficiency is determined by dwell time at misincorporated bases, not solely by selectivity for mismatches.
    • Results offer insights into T4 DNA polymerase structure and its interaction with accessory proteins for holoenzyme processivity.