Transcription-terminating lesions induced by bifunctional alkylating agents in vitro

The present study was initiated to determine if DNA damage induced by the bifunctional anti-tumor alkylating agents melphalan, nitrogen mustard, a spontaneously activated derivative of cyclophosphamide or chlorambucil inhibits transcription in vitro, and to determine if the potential sites of transcription termination correlate with the sites of N7 guanine adducts predominantly formed by these agents. To assess drug effects on in vitro transcription, linearized plasmid DNA containing the 420-bp PstI fragment of exon two of the human c-myc oncogene was incubated with various concentrations of the drugs. After drug removal and further drug-free incubation, the sense strand of the c-myc insert was transcribed with either of two bacteriophage RNA polymerases in the presence of [32P]UTP. The labeled products of the reaction were electrophoresed next to the labeled products of RNA sequencing reactions, and the location of transcription termination along the DNA template was determined. The sites of transcription termination were then compared with the sites of drug-induced guanine N7 alkylation in the template, as determined by modified Maxam-Gilbert sequencing. At the drug exposures examined, all the drugs were shown to alkylate any guanine in the template. Transcription of this alkylated DNA, however, resulted in RNA molecules truncated not at every alkylated guanine, but at various discrete sites throughout the template. Transcription was terminated at every adenine pair examined in the melphalan-treated template, at selected guanine pairs in the nitrogen-mustard-treated template, and at selected adenine-guanine and guanine-adenine pairs in the chlorambucil-treated template. Transcription of cyclophosphamide-derivative-treated DNA was unaffected. These results suggest that only some bifunctional alkylating agents induce DNA damage capable of terminating transcription in vitro, and that these agents do so in a sequence-specific, drug-specific manner inconsistent with patterns of guanine N7 alkylation.