Nucleas action on chromatin: evidence for discrete, repeated nucleoprotein units along chromatin fibrils.
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Abstract:
The time course of the fragmentation of calf thymus chromatin by DNase II (deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6) has been examined by sedimentation of chromatin digests through linear (5-20%) sucrose gradients. The action of nuclease is decidedly nonrandom, ultimately producing roughly equal amounts of acid-soluble oligonucleotides and 11S nucleoprotein particles. The 11S particles contain double-stranded DNA that is approximately 400 A or 120 base-pairs long, as measured by electron microscopic examination of deproteinized samples, and is maintained in a compact conformation within the intact particles. In addition, 15S nucleoprotein particles containing predominantly 800-A lengths of DNA have been isolated from less extensively digested chromatin. Evidence is presented which indicates that the 11S particles are fundamental structural units that are arranged in tandem along certain regions of chromatin fibrils. Preliminary experiments with different nucleases and with chromatin from different mammalian species indicate that these results are a natural consequence of the arrangement of DNA and proteins in mammalian chromatin and are not peculiar to the system described in detail.Keywords:
Nucleoprotein
Micrococcal nuclease
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Extracts of Drosophila embryos can mediate the assembly of a chromatinlike structure from histones and DNA under physiological conditions. The histone-DNA complex formed in vitro contains micrococcal nuclease-sensitive sites spaced at 200-base pair intervals. More extensive digestion of the complex by micrococcal nuclease generates 11S particles which cosediment with nucleosome core particles isolated from native chromatin. These particles contain 140-base pair DNA fragments which upon further cleavage with micrococcal nuclease give rise to a pattern of discretely sized DNA fragments characteristic of nucleosome core particles. We have assayed the chromatin assembly process both qualitatively by measuring the induction of supertwists into a relaxed circular DNA (a process requiring a nicking-closing enzyme) and quantitatively by measuring the formation of micrococcal nuclease-resistant DNA fragments from radioactively labeled linear DNA. The amount of chromatin formed depends primarily on the amount of histones, whereas the rate of assembly depends on the amount of extract protein added. The factors in the extract that mediate chromatin assembly appear to interact first with the DNA because preincubation of the DNA with the extract markedly increases the extent of assembly.
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Micrococcal nuclease
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We have developed a method of preparing yeast chromatin that facilitates the analysis of nucleoprotein organization. Yeast chromatin, isolated as an insoluble complex, is digested with micrococcal nuclease and fractionated into major insoluble and soluble fractions. Mo nucleosomal repeat is seen early in digestion for the insoluble fraction. Nucleosomal complexes of the soluble fraction are excised by nuclease in a distinctive non-random pattern; they are markedly depleted in mononucleosomes. When we analyze the soluble material by high resolution native electrophoresis, we find that the nucleoproteins resolve into two bands for each DNA multimer of the nucleosomal repeat. Our results suggest that there are structural similarities between bulk yeast chromatin and chromatin configurations found in transcribing genes of complex eukaryotes.
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We digested polyoma virus nucleoprotein complex, isolated from disrupted virions, with micrococcal nuclease and DNase I. The results were compared with digestions of chromatin from mouse nuclei. The nucleosome "core" structures were similar, but the spacing of the nucleosomes in the isolated polymoma nucleoprotein complexes was irregular, whereas in mouse chromatin it was regular. The average nucleosome repeat length in each case was 190 to 200 base pairs. This figure suggests that, unless there are substantial stretches of free DNA, the polyoma nucleoprotein complex contains about 26 nucleosomes. The commonly used method of preparing the nucleoprotein complex by disruption of virions at pH 10.2 may lead to significant damage to the structure. Such damage may be more clearly revealed by the susceptibility of the DNA to nuclease digestion than by the usual criteria of sedimentation velocity and buoyant density.
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Journal Article Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I Get access Gad Galili, Gad Galili Department of Plant Genetics, The Weizmann Institute of ScienceRehovot 76100, Israel Search for other works by this author on: Oxford Academic PubMed Google Scholar Abraham Levy, Abraham Levy Department of Plant Genetics, The Weizmann Institute of ScienceRehovot 76100, Israel Search for other works by this author on: Oxford Academic PubMed Google Scholar Karl M. Jakob Karl M. Jakob + Department of Plant Genetics, The Weizmann Institute of ScienceRehovot 76100, Israel +To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 9, Issue 16, 25 August 1981, Pages 3991–4005, https://doi.org/10.1093/nar/9.16.3991 Published: 25 August 1981 Article history Received: 13 April 1981 Published: 25 August 1981
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To analyze the structure of the replicating regions of simian virus 40 nucleoprotein complex (SV40 chromatin), photochemical binding of 8-methoxypsoralen (8-MOP) and changes in digestability with micrococcal nuclease were studied. 8-MOP bound preferentially to the linker DNA of nucleosomes and strongly inhibited nuclease digestion. Nuclease digestability of newly synthesized DNA in the replicating chromatin was markedly increased, but it was inhibited in the early time of nuclease reaction by photobinding of 8-MOP. The data suggest that the replicating regions of chromatin are more exposed than the bulk of mature chromatin.
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Digestion of calf thymus chromatin with micrococcal nuclease produces a mixture of apparently well defined nucleoprotein fragments which have been partially resolved by sedimentation on linear (5-20%) sucrose gradients. Sedimentation patterns reveal a predominant peak at the 11S position, three slower components, which have not previously been reported, at the 3.4S, 5.3S and 8.6S positions, and three faster components at the 17S, 22S and 26S positions. DNA isolated from the 3S to 12S region of gradients has been resolved on polyacrylamide gels into nine to ten discrete components ranging from 47 to 156 base pairs in length. A nearly identical pattern of small DNA products was obtained from chromatin digested in intact nuclei. These data suggest that chromatin contains either several types of subunits or predominently a single type of subunit which can be asymmetrically cleaved at any one of four or more sites.
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Micrococcal nuclease
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Micrococcal nuclease
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Single-stranded binding protein
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