On the mechanism of nucleosome assembly by histone chaperone NAP1

doi:10.1074/jbc.M511619200

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Papers In Press, published online ahead of print March 12, 2006
J. Biol. Chem, 10.1074/jbc.M511619200
Submitted on October 26, 2005
Accepted on March 12, 2006

On the mechanism of nucleosome assembly by histone chaperone NAP1

Jacek Mazurkiewicz, J. Felix Kepert, and Karsten Rippe

Molecular Biophysics Group, Kirchhoff-Institut für Physik, Heidelberg D-69120

Corresponding Author: Karsten.Rippe{at}kip.uni-heidelberg.de

The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments of 146 and 207 bp length containing the L. 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 fast (k₂ = (4.1 ± 3.5)· 10⁵ M^-1 s^-1) by 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 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 or 207 bp DNA and upon substitution of the canonical H2A histone with H2A.Z. The distinct nucleosome/hexasome ratios are 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.

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