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

tel-00413908, version 1 - 7 Sep 2009
in deciphering site specific changes in the conformation of nucleosomal DNA. DNase I
digestion of canonical nucleosomes gives a 10 bp repeat, typical for 601 nucleosomes,
indicative of minor groove of nucleosomal DNA facing towards the solution (lanes 2-4).
Incorporation of H2A Δ109 in the nucleosome showed no major structural perturbations.
However, subtle changes were observed in the vicinity of nucleosomal dyad (lanes 5-7).
Further deletion of C teminal residues, i.e H2A Δ97 which lacks α-C helix and H2A Δ90
which lacks all of the C-terminal tail as well as the last two α helices, results in clear
perturbation in the conformation of nucleosomal DNA (lanes 8-10, 11-13). Prominent
changes are indicated by the asterisk. Similar perturbations are also seen when all of H2A C-
terminal as well the docking domain is completely deleted (H2A Δ79) as seen in lanes 14-16
or replaced with docking domain of H2A.Bbd (lanes 17-19) leading to a DNase I digestion
profile quasi-identical to H2A.Bbd nucleosomes (lanes 20-22). Note that the N-terminal HA
tag on deletion proteins does not contribute to these changes as HA tagged conventional H2A
and untagged H2A containing nucleosomes exhibit identical DNase I digestion profile
(Compare lane 4 to 23).
In parallel, we performed OH° footprinting (Figure IV.2B) on the nucleosomes containing
H2A.Bbd (lane 2), H2A.ddBbd (lane 3), and H2A Δ79 (lane 4). A 10 base periodic repeat was
found similar to nucleosomes containing canonical H2A (lane 1) confirming the wrapping of
DNA around the histone octamer. This is not surprising as either type of nucleosomes may
not pose a steric hindrance towards OH° as seen with DNase I (Hayes and Lee, 1997).
An interesting phenomenon observed here is the progressive appearance of specific bands in
DNase I profile with progressive deletion of C-terminal region of H2A. As described before
the C terminal domain H2A perform two major functions (i) organisation of last turn of DNA
through interaction with H3 αN helix and (ii) formation of a β-sheet interaction with C-
terminal of H4, thus contributes to the strength of dimer-tetramer interaction (Luger et al.,
1997). Note that histone octamer is not stable at physiological salt conditions (Eickbush et al.,
1978). This is due to weak nature of interactions between H2A-H2B dimer and (H3-H4)2
tetramer and wrapping of DNA contributes significantly in maintaining the interaction
between the two (Luger et al., 1997; Bao et al., 2004). Therefore, the perturbations observed
deep inside the nucleosome by DNase I footprinting could be largely attributed to weakened
dimer-tetramer interactions. This weakening could be caused indirectly by (i) loss of
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