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Related Experiment Videos

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in

P Manivasakam1, J Aubrecht, S Sidhom

  • 1Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA.

Nucleic Acids Research
|December 1, 2001
PubMed
Summary

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Restriction enzymes enhance illegitimate DNA integration in mammalian cells by creating breaks that attract repair, but they reduce homologous recombination efficiency. This competition impacts DNA repair pathways.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Mammalian cells possess two primary DNA double-strand break repair pathways: illegitimate end-joining and homologous recombination.
  • Understanding factors influencing these pathways is crucial for gene therapy and genetic engineering.

Purpose of the Study:

  • To investigate the impact of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells.
  • To determine if restriction enzyme-induced DNA breaks influence the choice between illegitimate recombination and homologous recombination.

Main Methods:

  • Utilized plasmids with a neo(R) expression cassette for selection of illegitimate integration events.
  • Employed restriction enzymes (BamHI, BglII, EcoRI, KpnI) in co-transfection experiments with CHO wild-type and xrcc5 mutant cells.

Related Experiment Videos

  • Studied illegitimate and homologous integration in mouse embryonic stem (ES) cells using a modified plasmid containing the neo(R) gene flanked by HPRT exon 3.

    • Main Results:

    • Restriction enzymes significantly increased linearized plasmid integration efficiency (up to 5-fold) in CHO wild-type cells, but not in xrcc5 mutant cells.
    • In mouse ES cells, restriction enzymes increased illegitimate integration several-fold.
    • Conversely, restriction enzymes decreased the absolute frequency of homologous integration approximately 2-fold and the percentage of homologous integration by over 10-fold.

    Conclusions:

    • Restriction enzyme-induced DNA breaks preferentially promote illegitimate recombination (IR).
    • IR events compete with the homology search process, thereby reducing homologous integration frequency.
    • The xrcc5 gene product is implicated in the mechanism by which restriction enzymes affect DNA integration.