Cleavage of chromatin with methidiumpropyl-EDTA . iron(II).

Abstract

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(I) and micrococcal nuclease cleavage. MPE-Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some doublestranded) 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(H) 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(ll) will be very useful in the analysis of chromatin structure.With our current appreciation of the nucleosome as the fundamental unit of chromatin condensation (1-3), it has been pertinent to ask whether or not there is a functional requirement for a particular nucleosomal array. This aspect of chromatin structure has been most often expressed in the concept of specific nucleosome positioning (or "phasing") at a few or many loci of the eukaryotic genome, perhaps in a cell-, tissue-, or development-specific manner. Possible advantages of such positioning have been envisaged by many investigators, although no positive evidence for its actual functional importance in vivo has yet been presented. Numerous studies arguing for a specific or, conversely, for a random distribution of nucleosomes have been reported, and these have been reviewed (4-7). Many of these experiments have utilized micrococcal nuclease for generation of nucleosomal arrays. The DNA is purified subsequent to the nuclear digestion and the cleavage sites are mapped by reference to well-characterized restriction sites. Unfortunately, micrococcal nuclease has a marked sequence preference and introduces cleavages into purified DNA at quite specific and reproducible positions (8,9). In some cases these occur at exactly the same sites in chromatin, leading to uncertainty concerning which is chromatin specific and which is purely sequence specific....

Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin

“…Numerous probes with different degrees of specificity have been used in bulk chromatin analysis. At one extreme of this spectrum are some chemical probes, such as hy- droxyl radical (Tullius and Dombroski 1986) and MPE (Cartwright et al 1983), that show a modest degree of preferential cleavage relative to the nucleosome position, but which have little or no bias in terms of the underlying sequence. At an opposite end of the specificity spectrum are enzymatic probes, such as micrococcal nuclease (Wingert and Von Hippel 1968;Horz and Altenburger 1981) and the apoptotic nuclease DFF40 (Widlak et al 2000), which have some sequence bias, but which show a much stronger respect for nucleosome boundaries in their initial attack on a chromatin template.…”

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

“…S0(A-B-) was constructed as follows: CV20(A-) was partially digested with StuI ( Fig. 1 (8). Digestions were terminated by the addition of 0.1 volume of 50 mM bathophenanthrolinedisulfonate (Sigma Chemical Co.).…”

Nuclear Factor 1 and Octamer Transcription Factor 1 Binding Preset the Chromatin Structure of the Mouse Mammary Tumor Virus Promoter for Hormone Induction

“…NF1 and Oct1 Cooperatively Induce a Nucleosome Rearrangement in the MMTV LTR in the Absence of Hormoneactivated GR-The chemical nuclease MPE was used to monitor the nucleosome organization in the MMTV promoter, since it preferably cleaves internucleosomal linker DNA and lacks sequence-specific DNA cutting activity (12,13,39). By first injecting the single-stranded MMTV DNA reporter and then the various combinations of GR, NF1, and Oct1 mRNAs into pools of Xenopus oocytes, we were able to monitor the effects of NF1 and Oct1 on the chromatin structure.…”