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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM037677-04A1
Application #
3293189
Study Section
Molecular Biology Study Section (MBY)
Project Start
1986-12-01
Project End
1991-11-30
Budget Start
1990-12-01
Budget End
1991-11-30
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Hoepfner, R W; Sinden, R R (1993) Amplified primer extension assay for psoralen photoproducts provides a sensitive assay for a (CG)6TA(CG)2(TG)8 Z-DNA torsionally tuned probe: preferential psoralen photobinding to one strand of a B-Z junction. Biochemistry 32:7542-8
Jupe, E R; Sinden, R R; Cartwright, I L (1993) Stably maintained microdomain of localized unrestrained supercoiling at a Drosophila heat shock gene locus. EMBO J 12:1067-75
Ussery, D W; Sinden, R R (1993) Environmental influences on the in vivo level of intramolecular triplex DNA in Escherichia coli. Biochemistry 32:6206-13
Sinden, R R; Wells, R D (1992) DNA structure, mutations, and human genetic disease. Curr Opin Biotechnol 3:612-22
Zheng, G X; Kochel, T; Hoepfner, R W et al. (1991) Torsionally tuned cruciform and Z-DNA probes for measuring unrestrained supercoiling at specific sites in DNA of living cells. J Mol Biol 221:107-22
Kochel, T J; Sinden, R R (1989) Hyperreactivity of B-Z junctions to 4,5',8-trimethylpsoralen photobinding assayed by an exonuclease III/photoreversal mapping procedure. J Mol Biol 205:91-102
Esposito, F; Brankamp, R G; Sinden, R R (1988) DNA sequence specificity of 4,5',8-trimethylpsoralen cross-linking. Effect of neighboring bases on cross-linking the 5'-TA dinucleotide. J Biol Chem 263:11466-72
Kochel, T J; Sinden, R R (1988) Analysis of trimethylpsoralen photoreactivity to Z-DNA provides a general in vivo assay for Z-DNA: analysis of the hypersensitivity of (GT)n B-Z junctions. Biotechniques 6:532-43
Zheng, G X; Sinden, R R (1988) Effect of base composition at the center of inverted repeated DNA sequences on cruciform transitions in DNA. J Biol Chem 263:5356-61
Esposito, F; Sinden, R R (1987) Supercoiling in prokaryotic and eukaryotic DNA: changes in response to topological perturbation of plasmids in E. coli and SV40 in vitro, in nuclei and in CV-1 cells. Nucleic Acids Res 15:5105-24