Extrachromosomal recombination occurs efficiently in cells defective in various DNA repair systems.
A series of different frameshift mutations of a firefly luciferase reporter plasmid was created so that no activity was obtained when they were transfected into mammalian cells. Co-transfection of these constructs with short fragments of the original sequence resulted in luciferase activity in different cell lines (A-549, NIH 3T3 and Jurkat). The level of this activity was dependent on the length of the fragment, regardless of cell line examined. Two different transfection techniques (lipofection and adenovirus-enhanced gene transfer) gave similar results. It was shown by polymerase chain reaction that expression of detectable luciferase required recombination of the transfected molecules. Cells with defined defects in DNA repair pathways were examined for their ability to perform this extrachromosomal recombination. Cells lacking normal Ku p80, (ADP-ribosyl)transferase, MLH1 or XP-C were all capable of restoring expression to the frameshifted constructs. Given the pivotal roles of the above molecules in the pathways of DNA repair, it seems that this recombination derives from a different activity.
Morrison
C
C
Research Institute of Molecular Pathology, Vienna, Austria.
Wagner
E
E
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D002460:Cell Line; D004265:DNA Helicases; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D016368:Frameshift Mutation; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D000072200:Ku Autoantigen; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D016296:Mutagenesis; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000172:deficiency / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C088859:MLH1 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C501002:Xpc protein, mouse; D008156:Luciferases; D011065:Poly(ADP-ribose) Polymerases; D000070957:MutL Protein Homolog 1; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen