Etudes sur le mécanisme de remodelage des nucléosomes par ...

tel-00413908, version 1 - 7 Sep 2009
trinucleosome, a much larger structure than a single nucleosome was observed (Figure II.3D,
compare the structures of the trinucleosomes of the 2 nd row with those of the 3 rd row). The
linker DNA connecting this large structure with the adjacent nucleosomes was clearly visible
(Figure II.3D, 3 rd row). We attributed this structure to the RSC-single nucleosome complex.
Interestingly, this large structure exhibited a uniform staining, demonstrating that the
nucleosome indeed filled the RSC cavity (Figure II.3D, 3 rd row). This result is in agreement
with the recent cryo-EM data showing that RSC forms a complex with a single isolated
nucleosome (Chaban et al., 2008) and further illustrates that this is also the case when
nucleosomal arrays are used as substrate for the remodeler.
A C
B D
Figure II.3. Electron Cryo-Microscopy (EC-M) of the RSC treated mono- and trinucleosomes
shows that different species are present in the RSC remodeling reaction. (A) Centrally positioned
nucleosomes reconstituted on a 255 bp 601 DNA were treated with RSC for 30 minutes at 29°C in the
presence of ATP (under these conditions ∼ 30% of the nucleosomes were completely mobilized) and
then immediately vitrified. The first two rows show the nucleosomes exhibiting ‘standard’ structure,
i.e. non-mobilized nucleosomes (the first row) and completely mobilized nucleosomes (the second
row). The remaining four rows show the EC-M micrographs of the nucleosomes with altered structure.
Each micrograph is accompanied by schematic drawing illustrating the shape of the DNA observed in
the micrographs. (B) Incubation of 5S nucleosomes with RSC results in the generation of remosomes.
Centrally positioned nucleosomes were reconstituted on a 255 bp DNA fragment containing the 5S
somatic gene of Xenopus borealis. The 5S nucleosomes were treated with RSC as described in (A),
vitrified and visualized by cryo-EM. Non-affected (first row) and end-mobilized (second row) by
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