Nucleosome rearrangement in vitro. 1. Two phases of salt-induced nucleosome migration in nuclei

Watkins, John; Smerdon, Michael J.

doi:10.1021/bi00346a039

Cited by 19 publications

(12 citation statements)

References 45 publications

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“…These studies demonstrated the existence of two phases of salt-induced nucleosome rearrangement in nuclei in vitro (44). Following the first transition, due to histone octamer sliding, newly repaired DNA remains in an altered (or nonnucleosome) state (43).…”

Section: Discussionmentioning

confidence: 91%

Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

Baxter¹,

Smerdon

1998

Journal of Biological Chemistry

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The fate of nucleosomes during nucleotide excision repair is unclear. We have used organomercurial chromatography to capture accessible thiol groups of proteins at (or near) nascent repair sites in normal and xeroderma pigmentosum (group C) human cells. The reactive groups include cysteine 110 of histone H3, which is exposed in unfolded nucleosomes. Immediately after UV irradiation and a short pulse labeling of repair patches, intact nuclei were digested with restriction enzymes to release ϳ18% of the chromatin into soluble fragments, which are enriched (ϳ4-fold) in a constitutively transcribed gene. Upon organomercurial affinity fractionation, ϳ1.8% of the soluble chromatin remains bound in high salt (0.5 M NaCl) and is released with dithiothreitol. In normal cell chromatin, this fraction is enriched in nascent repair patches (1.5-1.8-fold) over the unbound fraction. This enrichment decreases following short chase periods with a time course similar to the loss of enhanced nuclease sensitivity of these regions (t 1 ⁄2 Ϸ 30 min). Much less enrichment of nascent repair patches is observed in the thiol-reactive fraction from XPC cells, which repair primarily the transcribed strand of active genes. These results suggest that transient nucleosome unfolding occurs during nucleotide excision repair in normal human cells, and this unfolding may require the XPC protein.

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Section: Discussionmentioning

confidence: 91%

Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

Baxter¹,

Smerdon

1998

Journal of Biological Chemistry

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“…However, the presence of bands corresponding to the nucleosome core and a compact dimer suggests that extensive sliding of the cores has taken place; the resulting randomization of nucleosome spacing would result in a smear after micrococcal nuclease digestion. It should be noted that transcriptionally active chromatin regions have been found to be depleted in HI (32) and that removal of HI facilitates sliding of nucleosome cores (33). The cause of the proposed core sliding could be the unwinding and overwinding of DNA due to the passage of the RNA polymerase complexes (34).…”

Section: Discussionmentioning

confidence: 99%

Chromatin structure of the developmentally regulated early histone genes of the sea urchinStrongylocentrotus purpuratus

1990

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Chromatin organization of the early histone gene repeat was studied at the early embryonic stages of the sea urchin S. purpuratus. Micrococcal nuclease digestion showed a highly irregular packaging of the whole repeat at the period of transcriptional activity, which was progressively replaced by more regular nucleosomal arrays upon developmentally programmed inactivation. No evidence for unique positioning of the nucleosomes was found. Regions upstream of each of the genes were hypersensitive to DNAase I digestion in the active state. These regions contained one (H2A and H2B), or two (H3 and H4) well-defined DNAase I cutting sites, or two poorly-defined sites (H1). They mapped within DNA sequences shown previously to be required for proper expression of the genes. Hypersensitivity continued in the hatching blastula, which have a conventional nucleosomal structure and a much reduced transcriptional activity. Hypersensitivity of these regions during morula and early blastula was not dependent on the torsional strain in chromatin, as it was not influenced by extensive gamma ray-induced nicking of the DNA in nuclei. By late blastula no hypersensitive regions were present.

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“…When done at 40C, NaCl extraction of H1 should produce little or no nucleosome sliding (9,10). If any sliding occurred during the extraction, it should not affect the results since it occurs before the formation of TDs.…”

Section: Methodsmentioning

confidence: 99%

“…Another interesting feature of Hi-containing nucleosomes is the absence of an obvious peak corresponding to positions +7 and -7 helical turns from the midpoint-i.e., 10 the ends. However, if a small peak were present near the 3' end, it might be obscured by the nearby artifactual peak due to undigested full-length molecules.…”

Section: Plot D)mentioning

confidence: 96%

Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin.

Pehrson

1989

Proc. Natl. Acad. Sci. U.S.A.

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Photo-induced thymine dimer formation was used to probe nucleosome structure in nuclei. The distribution of thymine dimers in the nucleosome and recent studies of the structure of thymine dimer-containing DNA suggest that the rate of thymine dimer formation is affected by the direction and degree of DNA bending. This premise was used to construct a model of the path of DNA in the nucleosome, which has the following features. (i) There are four regions of sharp bending, two which have been seen previously by x-ray crystallography of the core particle. (i) The DNA in Hi-containing nucleosomes deviates from its superhelical path near the midpoint; this is not seen with Hl-stripped chromatin. (ii) The internucleosomal (linker) DNA appears to be relatively straight.Much of the knowledge about nucleosome structure comes from studies of isolated nucleosome core particles. Such studies provide only limited information about native chromatin structure since these particles lack histone H1, the internucleosomal (linker) DNA, and any constraints imposed by higher order chromatin structure. Therefore, it is valuable to develop methods to study nucleosome structure in situ. One such method is photodimerization of pyrimidines. Recently, the distribution of pyrimidine dimers (PDs) in core particles isolated from irradiated chromatin was described in careful detail (1, 2). Peaks of PD formation were found to occur with an average periodicity of 10.3 bases and were interpreted in terms of a footprint of histone-DNA interactions. Recent reports indicate that DNA in the immediate vicinity of a thymine dimer (TD) is bent toward the major groove (3, 4). Therefore, TDs should form preferentially wherever the DNA is already bent toward the major groove, and their distribution might reveal a great deal about the path of DNA in the nucleosome. Very little is known about the effect of H1 on the path of DNA in the nucleosome or about the path of DNA in the internucleosomal (linker) region. As this information is important for understanding higher order chromatin structure and the role of H1 in nucleosome structure, I have explored the possibility of using TD formation as an approach to gain such information. (5) except for the peak at 4.726; instead they reported a peak at 2.246. Also, the melting point differs from the one they reported, 283-284TC. However, the NMR spectrum and elemental analysis given here are consistent with the structure of AcOD, and the compound was found to be effective in greatly stimulating the production of TDs in DNA irradiated with light >310 nm. MATERIALS AND METHODSIsolation of Nuclei. All steps were carried out at 0-40C unless otherwise stated. Frozen rat livers (Pel-Freez Biologicals) were homogenized with a Waring blender in buffer A [0.34 M mannitol/60 mM KCl/15 mM NaCl/0.15 mM spermine/0.5 mM spermidine/2 mM EDTA/0.5 mM EGTA/15 mM triethanolamine/1% thiodiglycol/0.25 mM phenylmethanesulfonyl fluoride, pH 7.3] and filtered through cheese cloth, and nuclei were isolated essentially as described (8...

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Nucleosome rearrangement in vitro. 1. Two phases of salt-induced nucleosome migration in nuclei

Cited by 19 publications

References 45 publications

Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

Chromatin structure of the developmentally regulated early histone genes of the sea urchinStrongylocentrotus purpuratus

Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin.

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