RESUMO
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.