Quantitative Agarose Gel Electrophoresis of Chromatin: Nucleosome-Dependent Changes in Charge, Shape, and Deformability at Low Ionic Strength

Terace M. Fletcher, Uma Krishnan, Philip Serwer, Jeffrey C. Hansen

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

The surface electrical charge density and the deformability of nucleosomal arrays have been characterized by quantitative agarose gel electrophoresis. Monodisperse linear DNA (2.5–3.3 kbp) was reconstituted with histone octamers into either saturated (∼1 nucleosome/200-bp DNA) or subsaturated (<1 nucleosome/200-bp DNA) nucleosomal arrays. The electrophoretic mobility (μ) of both nucleosome-free DNA and nucleosomal arrays was determined at low ionic strength in 0.2-3.0% agarose gels. A semilogarithmic plot of μ vs gel concentration was linear for DNA and convex for saturated nucleosomal arrays. By extrapolating the μ to 0% agarose, the magnitude of the gel-free μ of saturated nucleosomal arrays was found to be ∼20% lower than that of nucleosome-free DNA molecules. This difference is explained by the net neutralization of ∼85 DNA negative charges by each histone octamer. By using an internal standard to measure the effective pore size (Pe) of the agarose gel, the effective radius (R) of DNA and nucleosomal arrays was determined at each agarose concentration. In the more dilute gels (Pe ≥ 400 nm), the differences between the effective R values of DNA, subsaturated nucleosomal arrays, and saturated nucleosomal arrays are consistent with the differences in their hydrodynamic shapes as measured by analytical velocity centrifugation. However, as Pe decreases, the effective R of both nucleosome-free DNA and subsaturated nucleosomal arrays decreases significantly. This is in contrast to the effective R of saturated nucleosomal arrays, which remains constant at all Pe. The decrease in effective R of the DNA and subsaturated nucleosomal arrays is explained by conformational deformation (i.e., stretching) coupled with reptation during gel electrophoresis [Slater, G. W., Rousseau, J., Nooland, J., Turmel, C., & Lalande, M. (1988) Biopolymers 27, 509-524]. These data suggest that the deformability of a nucleosomal array at low ionic strength decreases abruptly as the level of saturation approaches 1 nucleosome per 200-bp DNA.

Original languageEnglish (US)
Pages (from-to)2226-2233
Number of pages8
JournalBiochemistry
Volume33
Issue number8
DOIs
StatePublished - Mar 1 1994

ASJC Scopus subject areas

  • Biochemistry

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