Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms.
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.
Liu
Yaobin
Y
Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX, USA.
Nairn
Rodney S
RS
Vasquez
Karen M
KM
D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D005363:Ficusin / Q000737:chemistry*; D005785:Gene Conversion; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D017384:Sequence Deletion
D003432:Cross-Linking Reagents; C051802:triplex DNA; D004247:DNA; D005363:Ficusin