DNA in both eukaryotic and prokaryotic chromosomes is organized into supercoiled topological domains. In prokarytotes about half the supercoils are equilibrated as unrestrained torsional tension in the DNA. Such strain has been shown to be important in mechanisms controlling gene expression. In eukaryotes, most supercoils are restrained by nucleosomes and,on average, the DNA helix is not wound with torsional tension. Although there is no direct evidence for unrestrained supercoiling in eukaryotic DNA, some current gene regulatory models suggest that a torsionally strained helix is involved. One model suggests that torsional tension in eukaryotic chromatin may be generated by the movement of RNA polymerase during transcription. Torsional tension could promote breathing the DNA, formation of cruciforms, formation of triple stranded DNA, or formation of left-handed Z-DNA. The proposal is designed to determine if torsional tension exists in eukaryotic DNA. Our origin in vivo assay for unrestrained torsional tension involved measurement of rates of trimethylpsoralen photobinding to DNA. This provided an averaging measurement which would not detect torsional tension if present in a small fraction of the chromosome, such as in active genes or at a specific localized site in the DNA. This assay will be modified to quantitate rates of psoralen cross-linking to selected restriction fragments of DNA to measure torsional strain within a selected region of DNA. We will synthesize, characterize, and apply cruciform and Z-DNA """"""""torsionally tuned probes"""""""" for unrestrained supercoiling in vivo. This involves application of psoralen-based assays we have developed that distinguish the conformation of potential cruciforms and Z-DNA sequences in living cells. These alternate helical structures from at precise minimum (tuned) superhelical densities allowing measurement of the level of supercoiling in vivo at specific sites in the DNA. The cross-linking assay will be applied in vivo to determine if selected regions of SV40 minichromosomes or various Drosophila genes are wound with unrestrained torsional tension in living cells. In addition we will insert the """"""""torsionally tuned probe"""""""" sequences into the control region of SV40 DNA, on either end of an inducible gene in a stable myc epichromosomal vector, and adjacent to Drosophila hsp70 heat shock genes to ask if unrestrained DNA supercoiling can be detected constitutively or in association with transcription in vivo.
