PRESENCE OF NEGATIVE TORSIONAL TENSION IN THE PROMOTER REGION OF THE TRANSCRIPTIONALLY POISED DIHYDROFOLATE-REDUCTASE GENE IN-VIVO

DNA topology has been suggested to play an important role in the process of transcription. Negative torsional tension has been shown to stimulate both pre-initiation complex formation and promoter clearance on plasmid DNA in vitro. We recently showed that genomic DNA in human cells contains localized torsional tension. In the present study we have further characterized and mapped torsional tension in the dihydrofotate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells and investigated the effects of differential rates of transcription on the magnitude and location of this tension. Using psoralen photo-cross-linking in conjunction with X-irradiation, we found that relaxable psoralen hypersensitivity was specifically localized to the promoter region of the serum-regulated DHFR gene in serum-stimulated, but not in serum-starved, cells. Moreover, this hypersensitivity did not appear to be caused by transcription elongation, since it persisted in cells in which transcription of the DHFR gene had been reduced by the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). We suggest that the generation of negative torsional tension in DNA may play an important role in gene regulation by poising genes for transcription.