On the mechanism of nucleosome assembly by histone chaperone NAP1.
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 k(1) = (2.5 +/- 0.7) . 10(4) m(-1) s(-1). The tetrasome was converted quickly (k(2) = (4.1 +/- 3.5) . 10(5) 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 k(3) = (6.6 +/- 1.4) . 10(3) 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 k(4) = (7.7 +/- 3.7) . 10(3) m(-1) s(-1). The activity of the chromatin remodeling complex ACF did not increase the kinetics of the mononucleosome assembly process.
Molecular Biophysics Group, Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany.
Jacek
J
Mazurkiewicz
J Felix
JF
Kepert
Karsten
K
Rippe
D016880:Anisotropy; D018797:Cell Cycle Proteins / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D004275:DNA, Ribosomal / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006657:Histones / Q000737:chemistry*; D007700:Kinetics; D047331:Lytechinus / Q000378:metabolism*; D008954:Models, Biological; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry; D009687:Nuclear Proteins / Q000378:metabolism*; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes / Q000378:metabolism*; D013997:Time Factors
D018797:Cell Cycle Proteins; D004275:DNA, Ribosomal; D006657:Histones; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes