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Figure 3.
Figure 3. Interaction of Histones H3 and H4 with RbAp46
(A) Analytical size-exclusion chromatography of the recombinant
histone H3/H4 complex used in the biochemical experiments
described in this paper. The inset panel shows a Coomassie
blue-stained 4%–12% NuPAGE gel used to analyze the fractions.
In the conditions used here (2 M NaCl and 20 mM HEPES [pH 7.5],
on a Superdex 75 PC3.2/30 column), histones H3 and H4 are
present as tetramers, but at lower ionic strengths (as used in
the binding experiments) these dissociate to form a mixture of
dimers and tetramers (Banks and Gloss, 2003).
(B) Pull-down
of either wild-type or mutant RbAp46 by histones H3 and H4
crosslinked to DynaBeads, in the absence or presence of the
N-terminal histone H4 peptide (residues 16–41). (The
experiments were carried out as described in Figure 2. The
positively charged lysozyme protein was also crosslinked to
beads in separate experiments and was used as a negative
control.)
(C) Comparison of the interactions of Ile-34,
Leu-37, and Ala-38 in helix 1 of histone H4 with (i) the
N-terminal helix of RbAp46 in the RbAp46/histone H4 peptide
structure, (ii) α helices 2 of histone H3 and H4 in one (of the
two) H3/H4 dimer in the nucleosome core particle (Davey et al.,
2002; PDB code: 1KX5), and (iii) α helices 2 of histone H3 and
H4 in the ASF1-histone H3/H4 complex ([English et al., 2006] and
[Natsume et al., 2007]; PDB code: 2HUE). In (i), (ii), and
(iii), the view is down the axis of helix 1 of histone H4.
Because similar contacts are made between histones H3 and H4 in
the complex with ASF1 and in both copies of histones H3 and H4
in the nucleosome core particle, it is likely that isolated
histones H3 and H4 also interact with each other in a similar
manner. Histone H4 is colored blue in all three structures,
whereas histone H3 is yellow in the nucleosome core particle and
pink in the ASF1 complex.
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