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Related Experiment Videos

Deletion errors generated during replication of CAG repeats.

L C Kroutil1, T A Kunkel

  • 1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.

Nucleic Acids Research
|August 14, 1999
PubMed
Summary
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Triplet repeat instability, linked to diseases, involves DNA polymerase deletions. These polymerases create mutations by misaligning triplet repeats, with proofreading offering limited protection.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Triplet repeat sequence instability is a known factor in hereditary neurological disorders and cancers.
  • This instability can manifest as deletions of triplet repeats during DNA replication by polymerases.

Purpose of the Study:

  • To investigate the mechanism of triplet repeat deletion during DNA replication by specific DNA polymerases.
  • To quantify the frequency of deletions versus substitutions at triplet repeat sequences.
  • To assess the role of proofreading activity in mitigating triplet repeat instability.

Main Methods:

  • Constructed M13mp2 DNA substrates containing (CAG)9 and (CAG)17 repeats with a TAG codon.
  • Copied these templates using DNA polymerase beta and exonuclease-deficient T7 DNA polymerase.

Related Experiment Videos

  • Scored errors as blue plaque Lac revertants and sequenced DNA to identify substitutions or deletions.

    • Main Results:

    • DNA polymerase beta and exonuclease-deficient T7 DNA polymerase generated deletions of 1–8 repeats.
    • These polymerases utilized misaligned template-primers, leading to deletions of 3 to 45 nucleotides.
    • Deletion frequencies significantly exceeded substitution frequencies, especially without error correction.
    • Proofreading-proficient T7 DNA polymerase showed 2- to 10-fold lower deletion frequencies.

    Conclusions:

    • Triplet repeat sequences are highly susceptible to mutation due to deletions.
    • Misaligned triplet repeat sequences are subject to polymerase proofreading, though with reduced efficiency.
    • This highlights the risk of triplet repeat expansion/contraction in disease pathogenesis.