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Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells.

X B Chang, J H Wilson

    Proceedings of the National Academy of Sciences of the United States of America
    |July 1, 1987
    PubMed
    Summary
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    Modifying DNA ends with dideoxynucleotides significantly reduces end joining, favoring homologous recombination. This finding offers a method to control random versus targeted DNA integration in mammalian cells.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Cell Biology

    Background:

    • Exogenous DNA integration in animal cells often occurs randomly, with homologous recombination being less frequent.
    • Free DNA ends are critical for both random and homologous recombination processes.
    • Understanding the influence of DNA ends on recombination is key to controlling integration outcomes.

    Purpose of the Study:

    • To investigate how specific DNA end modifications affect the competition between end joining and homologous recombination.
    • To determine if altering DNA ends can shift the balance towards targeted (homologous) recombination over random integration.

    Main Methods:

    • Constructed a linear simian virus 40 genome with terminal repeats as a model substrate.
    • Transfected the substrate into monkey cells to observe extrachromosomal recombination.

    Related Experiment Videos

  • Analyzed the ratio of end-join products (random integration analog) to homologous recombination products (targeted integration analog).
  • Tested various DNA end types (blunt, sticky, mismatched) and modified ends using dideoxynucleotides.

    • Main Results:

    • Different DNA end types (blunt, sticky, mismatched) did not alter the ratio of homologous recombination to end joining.
    • Addition of dideoxynucleotides to 3' hydroxyls decreased end joining by 5-6 fold relative to homologous recombination.
    • This modification effectively shifted the balance towards homologous recombination.

    Conclusions:

    • The frequency of DNA end joining can be modulated by altering the input DNA's terminal structures.
    • Modifying DNA ends, specifically with dideoxynucleotides, can decrease random integration relative to targeted integration.
    • This suggests a potential strategy for enhancing targeted DNA integration in mammalian cells.