Torsionally tuned cruciform and Z-DNA probes for measuring unrestrained supercoiling at specific sites in DNA of living cells.

We describe the development and application of "torsionally tuned" Z-DNA and cruciform probes for analyzing the level of unrestrained supercoiling at specific sites in the DNA of living cells. This approach is applicable for the analysis of dynamic differences in supercoiled DNA in different parts of plasmid, bacterial, or eukaryotic chromosomes. Using a psoralen-based assay, we have shown that the Z-DNA forming sequence (CG)6TA(CG)6, cloned into plasmid pUC8, exists as Z-DNA in 30 to 40% of plasmid molecules in wild-type Escherichia coli. This level suggested an in vivo superhelical density of sigma = -0.034 at the site of insertion in the plasmid. A higher level of Z-DNA found in cells deficient in topoisomerase I (topA10) suggested an in vivo superhelical density of sigma = -0.048. We have constructed a set of torsionally tuned inverted repeated DNA molecules which require different superhelical densities for cruciform formation. Using these inverted repeats and a crosslink assay for cruciforms, we present quantitative evidence for the existence of cruciforms in living E. coli cells. Cruciform formation was dependent on DNA supercoiling in vivo and on the location of the inverted repeat within a plasmid. In topA10 cells cruciforms were detected in less than 0.5% of plasmids when cloned into two different transcriptional units: the lacZ and CAT genes. However, when cloned outside a transcriptional unit, cruciforms were found at levels up to 50% in topA10 cells. More cruciforms were found upstream than downstream from divergent promoters in pBR322. From analysis of the fraction of different inverted repeats existing as cruciforms in vivo and the levels of supercoiling required for cruciform formation in vitro, we estimate in vivo superhelical densities of sigma = -0.034 and -0.041 for the EcoRI site of pUC8-based plasmids in wild-type and topA10 cells, respectively.

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.

An amplified primer extension assay has been developed for quantitatively mapping the sites of psoralen photoaddition to DNA. This assay was applied to a torsionally tuned Z-DNA-probe that was specifically designed for the primer extension assay. The torsionally tuned Z-DNA forming sequence, (CG)6TA(CG)2(TG)8, forms Z-DNA in vitro at negative superhelical density: sigma = -0.05. The internal 5'-TA dinucleotide was reactive to psoralen when it existed as B-DNA. Upon the formation of Z-DNA, the internal 5'-TA no longer photobound psoralen. The torsionally tuned sequence was synthesized as an EcoRI fragment such that, when Z-DNA formed, the central 5'-AATT of the EcoRI sites was part of the B-Z junctions. The 5'-AATT sequence was not reactive with psoralen when it existed as B-DNA. When the 5'-AATT sequence existed as a B-Z junction, one strand of each junction became hyperreactive to psoralen. The TT directly 5' to the B-DNA-Z-DNA junction photobound psoralen in a strand-specific fashion. Quantitation of the relative rate of psoralen photobinding to the internal 5'-TA and the 5'-AATT at the B-Z junctions provides relationships that are characteristic of the level of supercoiling in DNA.

Topoisomerase I is the predominant nuclear protein from avian erythrocytes that can be covalently linked to DNA.

Nuclear extracts of erythrocytes contain proteins which stably or possibly covalently bind to DNA. These proteins can be detected by an assay which was originally developed to quantify stable binding of topoisomerases to DNA [Trask, DiDonato & Muller (1984) EMBO J. 3, 671-676]. In this report, we show that the number of activities detected by this assay in crude extracts of nuclei is limited predominantly to various forms of topoisomerase I. One form, a 50 kDa protein, copurifies with histone H1. Western blotting experiments suggest that the 50 kDa topoisomerase exists in chromatin along with the 105 kDa form. In addition, the ratio between the high and low-Mr forms is relatively constant in erythrocytes and embryonic fibroblasts. These results imply that the multiple forms are not unique to one tissue setting.