How damaged is the biologically active subpopulation of transfected DNA?
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 double-strand 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.
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D007668:Kidney; D008433:Mathematics; D008957:Models, Genetic; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral; D004262:DNA Restriction Enzymes