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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Apr 16;93(8):3608–3612. doi: 10.1073/pnas.93.8.3608

Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases.

M Brenneman 1, F S Gimble 1, J H Wilson 1
PMCID: PMC39658  PMID: 8622983

Abstract

In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.

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Selected References

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