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Related Experiment Videos

Exonucleolytic proofreading of leading and lagging strand DNA replication errors.

J D Roberts1, D C Thomas, T A Kunkel

  • 1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.

Proceedings of the National Academy of Sciences of the United States of America
|April 15, 1991
PubMed
Summary

Exonucleolytic proofreading occurs during simian virus 40 DNA replication in human cells, ensuring high fidelity on both leading and lagging DNA strands during synthesis.

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • DNA replication fidelity is crucial for maintaining genomic integrity.
  • Exonucleolytic proofreading is a key mechanism for correcting DNA synthesis errors.
  • Simian virus 40 (SV40) DNA replication provides a model system for studying eukaryotic DNA replication.

Purpose of the Study:

  • To investigate the occurrence of exonucleolytic proofreading during SV40 origin-dependent DNA replication in human cell extracts.
  • To compare the fidelity of leading and lagging DNA strand synthesis.
  • To determine if proofreading mechanisms are active on both DNA strands during bidirectional replication.

Main Methods:

  • Utilized a fidelity assay employing M13mp vectors with a TGA codon in the lacZ alpha gene to detect single-base substitution errors.

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  • Introduced strand-specific errors by manipulating deoxynucleoside triphosphate (dNTP) concentrations.
  • Assessed error rates by analyzing revertant plaques under conditions of varying dNTP concentrations and deoxyguanosine monophosphate addition.

    • Main Results:

    • Increased dNTP concentrations or addition of deoxyguanosine monophosphate led to elevated error rates for specific mispairs (A.dCTP and T.dGTP).
    • These findings indicate the involvement of exonucleolytic proofreading during bidirectional DNA replication.
    • Base substitution error rates were found to be similar for both leading and lagging strand synthesis.

    Conclusions:

    • Exonucleolytic proofreading actively participates in maintaining DNA replication fidelity during SV40 replication in human cell extracts.
    • Both leading and lagging strands are subject to proofreading, suggesting a robust error-correction system.
    • The similar error rates for leading and lagging strands imply comparable fidelity mechanisms operate on both newly synthesized DNA strands.