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.
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