DNAase I-hypersensitive sites of chromatin

Abstract

During the last few years nucleases have been found to be effective tools for the dissection of the structure of chromatic. DNAase I (or pancreatic DNAase, E.C.3.1.4.5) is an endonuclease with little DNA-sequence specificity. When eucaryotic nuclei are partially digested with DNAase I, the resultant DNA fragments form a continuum of sizes, perceived as a uniform smear on agarose gels. Nonetheless, work with DNAase I has been of continuing interest since the finding of Weintraub and Groudine (Science 193, 848-856, 1976) that active chromatin is preferentially digested by this enzyme. The data now in hand indicate that a region of the chromatin, including the region of transcription and extending many kilobases beyond it, is generally very sensitive to DNAase I for a gene in the active state. In contrast with these broad patterns of differential DNAase I sensitivity, we have recently recognized and studied specific sites in chromatin that are even more sensitive to DNAase I, now referred to as DNAase l-hypersensitive sites. Such sites were first observed when the gel blot-hybridization technique of Southern was used to analyze the consequence of a very light digestion of Drosophila nuclei by DNAase I. Using a specific recombinant plasmid probe, we observed a series of discrete bands, indicating specific cleavage of the DNA in chromatin. Control experiments showed that no such bands were observed following digestion of naked DNA with DNAase I, demonstrating that the hypersensitive sites exist as a consequence of the chromatin structure. The pattern of bands obtained from chromatin was unique for each of five cloned Drosophila loci examined, suggesting that the sites recognized by DNAase I were specifically and nonuniformly distributed along the chromatin fiber (Wu et al., Cell 76, 797-806,1979). The smear of fragments seen as the digestion pattern for the genome as a whole apparently represents the sum of thousands of discrete patterns, rather than indicating a lack of specificity in the digestion of chromatin by DNAase I. A number of such nuclease-sensitive sites have now been mapped precisely with different approaches, most frequently an indirect end-labeling technique first applied by Nedospasov and Georgiev using micrococcal nuclease (BBRC 92, 532-539, 1980) and by Wu using DNAase I (Nature 286, 854-860,198O). Nuclei are digested lightly with the nuclease to introduce an average of one cut within a region of interest, bounded but not intersected by a particular restriction site. A hypothetical case is shown in the figure (A), in which we wish to examine the region between the two Barn sites. DNA is purified from the digested chromatin Minireviews