Chromatin reconstitution on small DNA rings. V. DNA thermal flexibility of single nucleosomes

Ali Hamiche, Ariel Prunell

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The thermal flexibility of DNA minicircles reconstituted with single nucleosomes was measured relative to the naked minicircles. The measurement used a new method based on the electrophoretic properties of these molecules, whose mobility strongly depended on the DNA writhe, either of the whole minicircle, when naked, or of the extranucleosomal loop, when reconstituted. The experiment was as follows. The DNA length was first increased by one base-pair (bp), and the correlative shift in mobility resulting from the altered DNA writhe was recorded. Second, the gel temperature was increased so that the former mobility was restored. Under these conditions, the untwisting of the thermally flexible DNA due to the temperature shift exactly compensates for the increase in the DNA mean twist number resulting from the one bp addition. The relative thermal flexibility was then calculated as the ratio between the increases in temperature measured for the naked and the reconstituted DNAs, respectively. The figure, 0.69 (±0.07), was used to derive the length of DNA in interaction with the histones, 109 (±25) bp. Such length was in good agreement with the mean value of 115 bp we have previously obtained from the distribution of the angles between DNAs at the entrance and exit of similar nucleosomes measured from high resolution electron microscopy. This consistence further reinforces our previous conclusion that minicircle-reconstituted nucleosomes, with 1.3(109/83) to 1.4(115/83) turns of superhelical DNA, show no crossing of entering and exiting DNAs when the loop is in its most probable configuration, and therefore, that these nucleosomes behave topologically as "single-turn" particles. The present data are also within the range of values, 50 to 100 bp of thermally rigid DNA per nucleosome, obtained by others for yeast plasmid chromatin, suggesting that the "single-turn" particle notion may be extended to this particular case of naturally-occurring H1-free chromatin. However, these data are quite different from the 230 bp figure derived from thermal measurements of reconstituted H1-free minichromosomes. It is proposed that nucleosome interactions occurring in this chromatin, but not in yeast chromatin, may be partly responsible for the discrepancy.

Original languageEnglish
Pages (from-to)327-337
Number of pages11
JournalJournal of Molecular Biology
Volume228
Issue number2
DOIs
Publication statusPublished - 20 Nov 1992
Externally publishedYes

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Nucleosomes
Chromatin
Hot Temperature
DNA
Base Pairing
Temperature
Yeasts
Superhelical DNA
Histones
Electron Microscopy
Plasmids
Gels

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Chromatin reconstitution on small DNA rings. V. DNA thermal flexibility of single nucleosomes. / Hamiche, Ali; Prunell, Ariel.

In: Journal of Molecular Biology, Vol. 228, No. 2, 20.11.1992, p. 327-337.

Research output: Contribution to journalArticle

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abstract = "The thermal flexibility of DNA minicircles reconstituted with single nucleosomes was measured relative to the naked minicircles. The measurement used a new method based on the electrophoretic properties of these molecules, whose mobility strongly depended on the DNA writhe, either of the whole minicircle, when naked, or of the extranucleosomal loop, when reconstituted. The experiment was as follows. The DNA length was first increased by one base-pair (bp), and the correlative shift in mobility resulting from the altered DNA writhe was recorded. Second, the gel temperature was increased so that the former mobility was restored. Under these conditions, the untwisting of the thermally flexible DNA due to the temperature shift exactly compensates for the increase in the DNA mean twist number resulting from the one bp addition. The relative thermal flexibility was then calculated as the ratio between the increases in temperature measured for the naked and the reconstituted DNAs, respectively. The figure, 0.69 (±0.07), was used to derive the length of DNA in interaction with the histones, 109 (±25) bp. Such length was in good agreement with the mean value of 115 bp we have previously obtained from the distribution of the angles between DNAs at the entrance and exit of similar nucleosomes measured from high resolution electron microscopy. This consistence further reinforces our previous conclusion that minicircle-reconstituted nucleosomes, with 1.3(109/83) to 1.4(115/83) turns of superhelical DNA, show no crossing of entering and exiting DNAs when the loop is in its most probable configuration, and therefore, that these nucleosomes behave topologically as {"}single-turn{"} particles. The present data are also within the range of values, 50 to 100 bp of thermally rigid DNA per nucleosome, obtained by others for yeast plasmid chromatin, suggesting that the {"}single-turn{"} particle notion may be extended to this particular case of naturally-occurring H1-free chromatin. However, these data are quite different from the 230 bp figure derived from thermal measurements of reconstituted H1-free minichromosomes. It is proposed that nucleosome interactions occurring in this chromatin, but not in yeast chromatin, may be partly responsible for the discrepancy.",
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