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J. Biol. Chem., Vol. 281, Issue 24, 16462-16472, June 16, 2006

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On the Mechanism of Nucleosome Assembly by Histone Chaperone NAP1^*

From the Molecular Biophysics Group, Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany

The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3·H4 was released forming a DNA-histone tetramer complex with a time constant of k₁ = (2.5 ± 0.7) · 10⁴ M^–1 s^–1. The tetrasome was converted quickly (k₂ = (4.1 ± 3.5) · 10⁵ M^–1 s^–1), by the addition of a single H2A·H2B dimer, into a "hexasome," i.e. a nucleosome lacking one H2A·H2B dimer. From this intermediate a nucleosome was formed by the addition of a second H2A·H2B dimer with an average rate constant k₃ = (6.6 ± 1.4) · 10³ M^–1 s^–1. For the back-reaction, significant differences were observed between the 146- and 207-bp DNA upon substitution of the canonical H2A histone with H2A.Z. The distinct nucleosome/hexasome ratios were reflected in the corresponding equilibrium dissociation constants and revealed some differences in nucleosome stability. In a fourth reaction, NAP1 mediated the binding of linker histone H1 to the nucleosome, completing the chromatosome structure with k₄ = (7.7 ± 3.7) · 10³ M^–1 s^–1. The activity of the chromatin remodeling complex ACF did not increase the kinetics of the mononucleosome assembly process.

Received for publication, October 26, 2005 , and in revised form, February 27, 2006.

^* This work was supported by the Volkswagen Foundation "Junior Research Groups at German Universities" program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed. Tel.: 49-6221-549270; Fax: 49-6221-549112; E-mail: Karsten.Rippe{at}kip.uni-heidelberg.de.

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