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 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.

Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere

“…On the basis of analysis of the alphoid tetO -HAC structure, we propose a mechanism for its de novo formation in human cells from a synthetic alphoid DNA array (Figure ), the initial steps of which may be general for all HACs developed from different alphoid DNA arrays. This mechanism is very similar to those proposed to describe the fate of exogenous DNA following its transfection into mammalian cells, a process known to be accompanied by a high frequency of structural rearrangements, including both degradation and generation of concatamers. , Stable transfection of cells is typically achieved by integration of multiple copies of input DNA into host chromosomes . Indeed, such integrants typically represent up to 80% of all clones selected during HAC formation. ,,,,,− …”

“…This mechanism is very similar to those proposed to describe the fate of exogenous DNA following its transfection into mammalian cells, a process known to be accompanied by a high frequency of structural rearrangements, including both degradation and generation of concatamers. 44,45 Stable transfection of cells is typically achieved by integration of multiple copies of input DNA into host 46 Indeed, such integrants typically represent up to 80% of all clones selected during HAC formation. 2,4,6,16,22,27−31 We suggest that during transfection, multiple alphoid tetO vector molecules penetrate some of the target cells.…”

Intermolecular Recombination Assay for Mammalian Cells that Produces Recombinants Carrying both Homologous and Nonhomologous Junctions

“…When considering the length of RmI sequences present in the recombinants, we found that there was a wide variation ranging from a few hundred base pairs to almost the entire 7,100-bp molecule, with a uniform distribution of the various lengths (data not shown). This suggests that if nonhomologous recombination is the result of end-to-end ligation as has been suggested (12,48), then the vast majority of parental molecules are subjected to degradation or breakage before ligation. Another consideration is that the homologous sequences of both parental molecules can be readily maintained in a nonhomologous recombinant.…”

“…The fact that cotransfection with circular molecules gives homologous recombinants with the same overall structure, as with one or both parental molecules being linear, suggests that double-strand breaks are part of the recombination mechanism. Wake et al (48) have evaluated that each 5 to 15 kbp of transfected DNA is subjected to a double-strand break. If these double-strand breaks are in or near the homologous sequences, this could lead to pairing between homologous sequences sharing an overlap, resulting in a junction of the type proposed by Lin and colleagues (21) and Wake and colleagues (49).…”

“…This has prompted several authors to propose models for homologous recombination involving a double-strand break (21,44,49). Double-strand breaks have also been reported to increase illegitimate recombination (6,12), which is thought to involve ligation of free ends (12,48).…”