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

tel-00413908, version 1 - 7 Sep 2009
IV.2.7 RSC mediated remodeling is similar to SWI/SNF on H2A.dockingdomain.Bbd
nucleosomes
Although SWI/SNF was not able to mobilize H2A.ddBbd nucleosomes, however, it was able
to induce structural perturbations in the nucleosomes seen clearly in the restriction enzyme
assay. The results of nucleosome sliding assays show that RSC is more sensitive to defects in
the docking domain of H2A (Figure IV.5). This raised the question whether the initial
remodeling process by RSC is also affected by these defects. To test this, we performed a
similar one pot restriction enzyme accessibility assay. H2A.ddBbd nucleosomes were
remodeled in presence of RSC and the accessibility of remodeled nucleosomes was assayed
as described previously. Note that the activity of RSC was normalized with SWI/SNF by
comparing its sliding activity on nucleosomes containing conventional H2A. A representative
experiment is shown in figure IV.8. It is clearly seen that the RSC action on H2A.ddBbd
nucleosomes gives rise to accessibility changes essentially similar to that of SWI/SNF (Figure
IV.8A, compare lanes 1-7 to 8-14). The results were further confirmed when a quantitation of
the accessibility of unremodeled and RSC remodeled nucleosomes was performed (Figure.
IV.8B). Accessibility at different super helical locations of unremodeled H2A.ddBbd
nucleosomes was compared to RSC remodeled nucleosomes at 16 minute time point of
HaeIII digestion. A 2-3 fold increase in accessibility at d2-d4 was seen, consistent with the
previous result of SWI/SNF mediated remodeling on these nucleosomes. As expected,
accessibility at d0 does not change significantly. Moreover, no decrease in accessibility at d7
and d6 was observed, rather remodeling by RSC results in a small increase of accessibility at
these super helical locations. We conclude that the first step of nucleosome remodeling by
RSC is affected by a defective docking domain of H2A.
151