We have analyzed the pattern of supercoil-dependent, single strand-specific nuclease cleavage sites across 11.6 kb (11.6 × 103 base-pairs) of cloned Drosophila melanogaster DNA from locus 67B1. This region contains coding sequences for the heat shock proteins hsp23, hsp26 and hsp28 as well as for a 1.6 kb developmentally regulated transcript (R). Two major sites are detected on digestion with S1 nuclease or mung bean nuclease. The most prominent site maps 100 base-pairs upstream of hsp26 in a very pyrimidine-rich region adjacent to a known region of chromatin DNAase I hypersensitivity. The other site is located approximately 800 base-pairs upstream of hsp28 in an area devoid of such chromatin-specific features. BAL31 nuclease produces a different array, with three to six strong cleavages located in the spacer DNA approx. 0.1 to 1.0 kb upstream of the DNAase I hypersensitive sites of hsp28, hsp23 and R. Thus, for each gene in the cluster a localized sequence sensitive to the winding state of the DNA is observed 5′ to the gene. However, there is no precise coincidence of any of the major sites sensitive to BAL31 nuclease in the supercoiled plasmid with the sequences sensitive to DNAase I in chromatin. While all of the enzymes utilized in this study have prominent single strand-specific endonucleolytic activity, it is clear that they recognize different variants in the DNA structure induced by supercoiling. At least two classes of DNA perturbation have been detected.
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