Abstract
Transient transfection has been widely used in many biological applications including gene regulation and DNA repair, but, so far, little attention has been paid to cellular responses induced by the transfected DNA. Here, we report that double-stranded (ds) DNA introduced into mammalian cells induced expression of a variety of genes involved in DNA damage signaling and DNA repair. The expression profile of the induced genes was highly dependent on the cell type, suggesting interactions between exogenous dsDNA and cellular proteins. Moreover, each cell line elicited a markedly different level of intrinsic cellular responses to the introduced dsDNA. Furthermore, the presence of single-stranded oligonucleotides or short duplexes consisting of two complementary oligonucleotides did not affect cellular response, indicating that the induction was highly dependent on the structure and length of exogenous DNA. The extent of induction of DNA damage, signaling and DNA repair activities correlated to episomal and chromosomal gene correction frequencies. In addition, the presented data indicate that the presence of exogenous dsDNA triggered a DNA damage response by activation of ATR (ataxia telangiectasia-Rad3-related) but not ATM (ataxia telangiectasia mutated) pathway.
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References
Igoucheva O, Alexeev V, Yoon K . Oligonucleotide-directed mutagenesis and targeted gene correction: a mechanistic point of view. Curr Mol Med 2004; 4: 445–463.
Kren BT, Steer CJ . The application of DNA repair vectors to gene therapy. Curr Opin Biotechnol 2002; 13: 473–481.
Liu L, Parekh-Olmedo H, Kmiec EB . The development and regulation of gene repair. Nat Rev Genet 2003; 4: 679–689.
Igoucheva O, Alexeev V, Yoon K . Mechanism of gene repair open for discussion. Oligonucleotides 2004; 14: 311–322.
Andersen MS, Sorensen CB, Bolund L, Jensen TG . Mechanisms underlying targeted gene correction using chimeric RNA–DNA and single-stranded DNA oligonucleotides. J Mol Med 2002; 80: 770–781.
Lin FL, Sperle K, Sternberg N . Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products. Mol Cell Biol 1990; 10: 103–112.
Smith CL, Hager GL . Transcriptional regulation of mammalian genes in vivo. A tale of two templates. J Biol Chem 1997; 272: 27493–27496.
Igoucheva O, Peritz AE, Levy D, Yoon K . A sequence-specific gene correction by an RNA–DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system. Gene Therapy 1999; 6: 1960–1971.
Igoucheva O, Alexeev V, Yoon K . Targeted gene correction by small single-stranded oligonucleotides in mammalian cells. Gene Therapy 2001; 8: 391–399.
Igoucheva O, Alexeev V, Pryce M, Yoon K . Transcription affects formation and processing of intermediates in oligonucleotide-mediated gene alteration. Nucleic Acids Res 2003; 31: 2659–2670.
Razzaque A, Mizusawa H, Seidman MM . Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc Natl Acad Sci USA 1983; 80: 3010–3014.
Calos MP, Lebkowski JS, Botchan MR . High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci USA 1983; 80: 3015–3019.
Zhou BB, Elledge SJ . The DNA damage response: putting checkpoints in perspective. Nature 2000; 408: 433–439.
Osborn AJ, Elledge SJ, Zou L . Checking on the fork: the DNA-replication stress-response pathway. Trends Cell Biol 2002; 12: 509–516.
Valerie K, Povirk LF . Regulation and mechanisms of mammalian double-strand break repair. Oncogene 2003; 22: 5792–5812.
Williams KJ, Telfer BA, Airley RE, Peters HP, Sheridan MR, van der Kogel AJ et al. A protective role for HIF-1 in response to redox manipulation and glucose deprivation: implications for tumorigenesis. Oncogene 2002; 21: 282–290.
Bertrand P, Lambert S, Joubert C, Lopez BS . Overexpression of mammalian Rad51 does not stimulate tumorigenesis while a dominant-negative Rad51 affects centrosome fragmentation, ploidy and stimulates tumorigenesis, in p53-defective CHO cells. Oncogene 2003; 22: 7587–7592.
Jasin M . Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet 1996; 12: 224–228.
Bibikova M, Beumer K, Trautman JK, Carroll D . Enhancing gene targeting with designed zinc finger nucleases. Science 2003; 300: 764.
Porteus MH, Baltimore D . Chimeric nucleases stimulate gene targeting in human cells. Science 2003; 300: 763.
Acknowledgements
This work was supported in part by grants from the National Institute of Health (AR38923, CA72765) to KY and the Dermatology Foundation Career Development to VA.
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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)
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Igoucheva, O., Alexeev, V. & Yoon, K. Differential cellular responses to exogenous DNA in mammalian cells and its effect on oligonucleotide-directed gene modification. Gene Ther 13, 266–275 (2006). https://doi.org/10.1038/sj.gt.3302643
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DOI: https://doi.org/10.1038/sj.gt.3302643
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