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

tel-00413908, version 1 - 7 Sep 2009
Similar to the sliding assay conditions, in this AFM experiment, the length of the 601 DNA
used for reconstitution was 255 bp and the histone octamer was centrally positioned relative
to the ends of DNA, having a longer free DNA arm L+=56 bp and a shorter one L-= 52 bp.
For ΔL distribution, ΔL was calculated as ΔL= (L+ - L-)/2. For the LC distribution, Lc was
calculated as Lc=Lt –L+ - L-, where Lt is the total length of the 601 DNA used for
reconstitution.
As seen, the control (incubated in absence of ATP) as well as remodeled H2A.ddBbd
nucleosomes exhibited a wide ΔL peak distribution (in contrast to conventional nucleosomes,
see figure III.3C). Importantly, it does not change significantly by action of SWI/SNF,
confirming SWI/SNF is unable to mobilize these particles to the end of the DNA (ΔL=60bp)
(Figure IV.4 A), consistent with the results obtained from nucleosome sliding assays. The Lc
distribution profile of control nucleosomes shows a peak value at ~130 bp meaning that only
130 bp of DNA is attached to the histone octamer. Similar values were obtained in an AFM
study on H2A.Bbd nucleosomes (Montel et al., 2007) further emphasizing the role of docking
domain of H2A.Bbd in open structure of these nucleosomes. The remodeled H2A.ddBbd
nucleosomes, however, show an increase in DNA complexed length (mean value ~145 bp)
indicating towards structural perturbations imparted by SWI/SNF (Figure IV.4B). Note that
the Lc distribution of remodeled nucleosomes is very wide. This strongly indicates
fluctuations of linker DNA arms and suggests that the action of SWI/SNF on H2A.ddBbd
nucleosomes results in pumping of linker DNA inside the nucleosome and that the interaction
of the DNA to the octamer remains dynamic.
The data from nucleosome sliding assays and AFM analysis, taken together, proves that the
docking domain of H2A is required for nucleosome mobilization mediated through SWI/SNF.
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