Quantitative agarose gel electrophoresis of chromatin: nucleosome-dependent changes in charge, sharp, and deformability at low ionic strength.
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 (approximately 1 nucleosome/200-bp DNA) or subsaturated (< 1 nucleosome/200-bp DNA) nucleosomal arrays. The electrophoretic mobility (mu) 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 mu vs gel concentration was linear for DNA and convex for saturated nucleosomal arrays. By extrapolating the mu to 0% agarose, the magnitude of the gel-free mu of saturated nucleosomal arrays was found to be approximately 20% lower than that of nucleosome-free DNA molecules. This difference is explained by the net neutralization of approximately 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 > or = 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Fletcher
T M
TM
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760.
Krishnan
U
U
Serwer
P
P
Hansen
J C
JC
D000818:Animals; D002645:Chickens; D002843:Chromatin / Q000737:chemistry* / Q000302:isolation & purification; D004247:DNA / Q000737:chemistry; D004563:Electrochemistry; D004587:Electrophoresis, Agar Gel; D009707:Nucleosomes / Q000737:chemistry*; D009994:Osmolar Concentration; D011487:Protein Conformation
D002843:Chromatin; D009707:Nucleosomes; D004247:DNA