Nucleic Acids Research Advance Access originally published online on July 15, 2008
Nucleic Acids Research 2008 36(14):4680-4688; doi:10.1093/nar/gkn438
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Nucleic Acids Research, 2008, Vol. 36, No. 14 4680-4688
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms
Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX, USA
*To whom correspondence should be addressed. Tel: + 1 512 237 9324; Fax: +1 512 237 2475; Email: kvasquez{at}mdanderson.org
Received May 7, 2008. Revised June 24, 2008. Accepted June 25, 2008.
Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells.
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  Y. Liu, R. S. Nairn, and K. M. Vasquez Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells Nucleic Acids Res., October 1, 2009; 37(19): 6378 - 6388. [Abstract] [Full Text] [PDF]
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