DNA interstrand cross-links promote chromosomal integration of a selected gene in human cells

We have used integrative pSV2 plasmids to learn how DNA lesions affect nonhomologous recombination with human chromosomes. Enhanced stable transformation of fibrosarcoma cells with a selectable gene was observed after chemical modification of the plasmid DNA; thus, cells transfected with plasmid pSV2-gpt carrying photoadducts of the cross-linking agent 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) yielded four- to sevenfold-higher levels of Gpt+ transformants than were obtained with untreated plasmid. The enhancement due to HMT interstrand cross-links was at least as great as that due to the monoadducts. DNA hybridization analysis indicated that the enhanced transformation frequency resulted from an increased number of cells carrying integrated plasmid sequences rather than from a higher copy number per transformant. The enhancement was not seen with a plasmid missing the sequences flanking the minimal simian virus 40 gpt transcription unit. Cotransfection with untreated and HMT-treated plasmids suggested that the HMT-containing DNA interacted preferentially with some cellular factor that promoted chromosomal integration of the plasmid DNA. It is concluded that (i) interstrand cross-linking as well as intrastrand DNA adducts promote nonhomologous recombination in human chromatin and (ii) DNA sequences flanking the selectable genes are the targets for such recombinational events.