Cartwright, Iain L. and Hertzberg, Robert P. and Dervan, Peter B. and Elgin, Sarah C. R. (1983) Cleavage of chromatin with methidiumpropyl-EDTA·iron(II). Proceedings of the National Academy of Sciences of the United States of America, 80 (11). pp. 3213-3217. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:CARpnas83
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Methidiumpropyl-EDTA·iron(II) [MPE·Fe(II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE·Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE·Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE·Fe(II) and micrococcal nuclease cleavage. MPE·Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE·Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE·Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE·Fe(II) will be very useful in the analysis of chromatin structure.
|Additional Information:||Copyright © 1983 by the National Academy of Sciences. Communicated by J. L. Beauchamp, February 22, 1983. We gratefully acknowledge the gift of recombinant plasmids from the laboratories of D. Brutlag (mDmlO7), W. Gehring (12D1), and D. Hogness (cDm500). This work was supported by National Institutes of Health grants to S.C.R.E. (GM 30273) and P.B.D. (GM 27681) and a National Research Service Award (GM 07616) to R.P.H. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||Drosophila DNA, intercalation, micrococcal nuclease, DNA structure, nucleosome positioning|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||19 Jan 2006|
|Last Modified:||26 Dec 2012 08:44|
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