Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells.
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 neo(R) 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 neo(R) 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 approximately 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.
Manivasakam
P
P
Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA.
Aubrecht
J
J
Sidhom
S
S
Schiestl
R H
RH
D000818:Animals; D034961:Antigens, Nuclear; D001426:Bacterial Proteins; D016466:CHO Cells; D006224:Cricetinae; D004265:DNA Helicases; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology; D015245:Deoxyribonuclease BamHI / Q000502:physiology; D015246:Deoxyribonuclease EcoRI / Q000502:physiology; D015252:Deoxyribonucleases, Type II Site-Specific / Q000502:physiology; D018390:Gene Targeting; D000072200:Ku Autoantigen; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection
D034961:Antigens, Nuclear; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606512:Xrcc5 protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen