Abstract
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.
Highlights
Simple mixing of histones and DNA leads mainly to large, insoluble aggregates [18, 19]
nucleosome assembly protein 1 (NAP1) is generally regarded as a carrier for the H2A1⁄7H2B dimer, it interacts with similar affinity with H31⁄7H4 in vitro [32, 35, 36] and has been used as sole histone carrier for chromatin reconstitution with the chromatin assembly/remodeling factor ACF [37]
A tetrasome consisting of an (H31⁄7H4)2 tetramer is formed on the DNA followed by the addition of two H2A1⁄7H2B dimers
Summary
Simple mixing of histones and DNA leads mainly to large, insoluble aggregates [18, 19]. It is shown that NAP1-guided assembly includes an additional intermediate step, as after tetrasome deposition hexasomes are formed, which maturate into complete nucleosomes. NAP1-mediated Mononucleosome Assembly Proceeds via a Hexasome Intermediate—To investigate the kinetics of nucleosome assembly by histone chaperone NAP1, products and intermediates of the reaction were analyzed on agarose gels (Fig. 1).
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