LOCALIZED TORSIONAL TENSION IN THE DNA OF HUMAN-CELLS

Torsional tension in DNA may be both a prerequisite for the efficient initiation of transcription and a consequence of the transcription process itself with the generation of positive torsional tension in front of the RNA polymerase and negative torsional tension behind it. To examine torsional tension in specific regions of genomic DNA in vivo, we developed an assay using photoactivated psoralen as a probe for unconstrained DNA superhelicity and x-rays as a means to relax DNA. Psoralen intercalates more readily into DNA underwound by negative torsional tension than into relaxed DNA, and it can form interstrand DNA cross-links upon UVA irradiation. By comparing the amount of psoralen-induced DNA cross-links in cells irradiated with x-rays either before or after the psoralen treatment, we examined the topological state of the DNA in specific regions of the genome in cultured human 6A3 cells. We found that although no net torsional tension was detected in the bulk of the genome, localized tension was prominent in the DNA of two active genes. Negative torsional tension was found in the 5' end of the amplified dihydrofolate reductase gene and in a region near the 5' end of the 45S rRNA transcription unit, whereas a low level of positive torsional tension was found in a region near the 3' end of the dihydrofolate reductase gene. These results document an intragenomic heterogeneity of DNA torsional tension and lend support to the twin supercoiled domain model for transcription in the genome of intact human cells.