Abstract
Random integration is a phenomenon in which transfected DNA molecules integrate into (random sites of) the host genome via non-homologous recombination. Although it is assumed that repair of DNA double-strand breaks leads to random integration events, how these endogenous DNA lesions are generated in living cells is poorly understood. In this study, we present evidence that DNA topoisomerase IIa (Top2α) and reactive oxygen species (ROS) are responsible for causing genomic DNA damage that leads to random integration. Specifically, we employed a human pre-B lymphocyte cell line to examine the effects of cellular Top2 expression levels and oxygen concentrations during cell culture. We find that treating cells with Top2α siRNA significantly reduces random integration frequency, while the absence of Top2β had little or no impact. We also show that cells continuously cultured under low (3%) oxygen culture conditions after electroporation display reduced random integration frequency compared to that under normal (21%) oxygen conditions. These findings support the notion that Top2α protein and ROS are endogenous factors that can produce DNA damage leading to random integration of transfected DNA in human cells.
Highlights
Mammalian cells possess the ability to perform nonhomologous recombination reactions, which require little or no sequence homology between DNA substrates [1,2]
We further examined the contribution of Top2α by constructing a TOP2B-/-TOP2A+/- cell line by gene targeting (Figure S1), which led us find that the heterozygous disruption of TOP2A does not affect the random integration frequency even in the complete absence of Top2β (Figure 1A)
It has been generally assumed that random integration of foreign DNA results from the repair of DNA damage, spontaneous chromosomal double-strand breaks (DSBs) that are caused by endogenous factors
Summary
Mammalian cells possess the ability to perform nonhomologous recombination reactions, which require little or no sequence homology between DNA substrates [1,2]. NHEJ and homologous recombination are the two major pathways for repairing DNA double-strand breaks (DSBs) that result from endogenous mechanisms as well as exposure to exogenous genotoxic agents [4]. A practical application of nonhomologous recombination is the generation of transfectants (i.e., random integrants) that stably express a transgene(s) of interest. The precise mechanism of random integration is not fully understood, it is believed that random integration results from non-homologous recombination-mediated repair, NHEJ, of a spontaneous chromosomal DSB accidentally induced by endogenous factors [5,6,7]. We focused on DNA topoisomerase II (Top2) and reactive oxygen species (ROS) as the endogenous factors that induce DSBs causative of random integration
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