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

tel-00413908, version 1 - 7 Sep 2009
pumping towards the center of 15-20 bp DNA from each individual linker, which is
accompanied with extensive perturbation in the histone-DNA interactions. This results in the
formation of a multitude of nucleosome-like particles, termed remosomes, which contain 175-
180 bp DNA associated with the histones. During the second step, the SWI/SNF acts as a true
translocase by pumping and releasing DNA in one direction
III.2 Results
III.2.1 The initial step of SWI/SNF nucleosome mobilization mechanism is the
perturbation of the histone-DNA interactions and the generation of a non-mobilized
nucleosome-like particle associated with ∼180 bp of DNA.
By using AFM it was recently shown that during the SWI/SNF nucleosome remodeling
reaction, in addition to both the initial non-slided nucleosomes (associated with ∼150 bp of
DNA) and the completely slided nucleosomes, a third group of particles was observed, which
consisted of non-mobilized nucleosome-like particles, but associated with ∼175-180 bp of
DNA (Montel et al., 2007). The presence of the additional 30-35 bp associated with the
histone octamer suggests that the histone-DNA interactions within these non-mobilized
nucleosome-like particles might be perturbed. To test this, we used DNase I footprinting.
Briefly, we reconstituted centrally positioned nucleosomes by using highly purified
recombinant histones and 255 bp 601.1 DNA. Under the conditions used the efficiency of
reconstitution was very high (essentially no free DNA was observed in the reconstituted
samples) and the reconstituted particles exhibited the typical nucleosomal organization. The
centrally positioned 32 P-end labeled nucleosomes were incubated with different amounts of
SWI/SNF at 29°C with in the presence of ATP, the reaction was arrested with apyrase and
run on a 5% native PAGE (Figure III.1).
118