Nucleic Acids Research, 2001, Vol. 29, No. 23 4826-4833
© 2001 Oxford University Press
Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells
Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA
Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neoR expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neoR gene flanked by exon 3 of Hprt. The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration 
2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.
* To whom correspondence should be addressed at present address: Departments of Pathology and Environmental Health, UCLA School of Medicine and Public Health, 650 Charles E. Young Drive South, 71-295 CHS, Los Angeles, CA 90095, USA. Tel: +1 310 267 2087; Fax: +1 310 267 2578; Email: rschiestl{at}mednet.ucla.edu Present addresses:Palaniyandi Manivasakam, CombinatoRx, 650 Albany Street, Boston, MA 02118, USAJiri Aubrecht, Pfizer Global Research and Development, Groton, CT 06340, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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