How damaged is the biologically active subpopulation of transfected DNA?

Abstract

Relatively little is known about the damage suffered by transfected DNA molecules during their journey from outside the cell into the nucleus. To follow selectively the minor subpopulation that completes this journey, we devised a genetic approach using simian virus 40 DNA transfected with DEAE-dextran. We investigated this active subpopulation in three ways: (i) by assaying reciprocal pairs of mutant linear dimers which differed only in the arrangement of two mutant genomes; (ii) by assaying a series of wild-type oligomers which ranged from 1.1 to 2.0 simian virus 40 genomes in length; and (iii) by assaying linear monomers of simian virus 40 which were cleaved within a nonessential region to leave either sticky, blunt, or mismatched ends. We conclude from these studies that transfected DNA molecules in the active subpopulation are moderately damaged by fragmentation and modification of ends. As a whole, the active subpopulation suffers about one break per 5 to 15 kilobases, and about 15 to 20% of the molecules have one or both ends modified. Our analysis of fragmentation is consistent with the random introduction of doublestrand breaks, whose cause and exact nature are unknown. Our analysis of end modification indicated that the most prevalent form of damage involved deletion or addition of less than 25 base pairs. In addition we demonstrated directly that the efficiencies of joining sticky, blunt, or mismatched ends are identical, verifying the apparent ability of cells to join nearly any two DNA ends and suggesting that the efficiency of joining approaches 100%. The design of these experiments ensured that the detected damage preceded viral replication and thus should be common to all DNAs transfected with DEAE-dextran and not specific for viral DNA. These measurements of damage within transfected DNA have important consequences for studies of homologous and nonhomologous recombination in somatic cells as is discussed.Transfection of foreign DNA into somatic cells in culture has become an extremely important tool in modem molecular genetics, permitting studies of gene function

Illegitimate DNA integration in mammalian cells

“…[27][28][29][30][31] Transfected DNA can also be mutated at high frequency (on the order of 1%) by homology-independent means including point mutations, deletions, and more complex rearrangements such as insertion of genomic DNA. [32][33][34] (see also our unpublished work). Linear extrachromosomal DNA can also be efficiently circularized in the nucleus, and different molecules can be joined together to form concatamers 35 by processes referred to as nonhomologous end joining (NHEJ).…”

“…NHEJ often involves short sequence homology between the joined ends, and additions or deletions of approximately 25 nucleotides or less at the junctions. 34,36,37 Similar processes have been shown to occur during chromosomal double-strand break (DSB) repair, 27,29,38 which again suggests that extrachromosomal and chromosomal repair are mediated by similar cellular machinery.…”

Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

“…1) is adjacent to and outside the marker. The choice of the site eliminated the production of those small deletion mutants which would arise from "nibbling" of the ends (30). Linearization of the pAL11 plasmid before transfection produced substantial enhancement of the frequency of both recombined and deletion mutant plasmids in the progeny plasmid population.…”

“…The importance of double-strand breaks as an initiating event for recombination has been emphasized in recent models of recombination pathways (15,27), and it seems likely that recombination between homologous sequences on plasmids introduced into * Corresponding author. cells as superhelical templates is initiated by endonucleaselike activities in vivo (30).…”

Theoretical mechanisms in targeted and random integration of transgene DNA

“…Of the DNA that survives this journey, a substantial proportion sustains some degree of endonucleolytic or exonucleolytic damage. In contrast, virtually no damage occurs to DNA delivered directly into the nucleus by microinjection [22,47].…”