DNase I sensitivity

Vidali, G., Boffa, L.C., Bradbury, E.M., and Allfrey, V.G. (1978) Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H3 and H4 and increased DNase I sensitivity of the associated DNA sequences. Proc. Natl. Acad. Sci. 75(5), 2239-2243. 

Weisbrod S, Weintraub H (1979) Isolation of subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin. Proc Natl Acad Sci USA 76 630-635... 

Wood, W.I. and Felsenfeld, G. (1982) Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II. J. Biol. Chem. 10 257(13), 7730-7736. 

DNA are readily accessible to RNA polymerase binding and transcription. By contrast, most DNA in eukaryotic cells exists in a condensed form (chromatin), which is not readily accessible to transcription. The small fraction of DNA accessible to the RNA polymerase in any given cell type is especially sensitive to cleavage by mild treatment with bovine pancreatic DNase I. These regions of the DNA often contain bound RNA polymerase, modified histones, and additional nonhistone proteins. Active regions are often undermethylated compared with the total DNA. Most of the methylated groups in DNA are on the C residues in the CG sequence. 

Unless otherwise noted, all procedures should be performed at 4°C or on ice. Once prepared. Drosophila nuclei should be handled identically, regardless of source. The nucleus-enriched pellet should be resuspended in 20 mM Tris-HCl, pH 7.5,5 mM MgCla (Buffer B). For nuclear resuspension, one volume of Buffer B relative to the initial volume of starting material should be used. For example, if nuclei are purified from 10 ml of embryos, 10 ml of Buffer B should be used if nuclei are purified from 1 ml of tissue culture cells, 1 ml of Buffer B should be used. Complete resuspension should be assured by Dounce homogenization (two-four strokes, loose pestle) and both DNase I and RNase A should be added to final concentrations of 10 and 8 /i.g/ml, respectively. Typically, RNase A is added first, the sample is mixed on a vortex mixer, and then the DNase I is added. After addition of DNase I, vortex mixing is avoided because DNase I is very sensitive to inactivation by oxidation. Rather, the sample is mixed by gentle agitation. 

Because DNase I is extremely sensitive to inactivation by proteases in the absence of Ca, buffers routinely contain protease inhibitors such as DFP and PMSC. Otherwise DNase I is very stable, retaining full activity for more than 10 days at pH 8 and 37°C. 

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