Cleavage of chromatin with methidiumpropyl-EDTA . iron(II).
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.
D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects; D002843:Chromatin / Q000187:drug effects* / Q000502:physiology; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006657:Histones / Q000302:isolation & purification; D008970:Molecular Weight
D002843:Chromatin; D006657:Histones; C038200:methidiumpropyl-EDTA-iron(II); D004247:DNA; D004492:Edetic Acid