Etudes sur le mécanisme de remodelage des nucléosomes par ...
Etudes sur le mécanisme de remodelage des nucléosomes par ...
Etudes sur le mécanisme de remodelage des nucléosomes par ...
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tel-00413908, version 1 - 7 Sep 2009<br />
HaeIII<br />
time<br />
A B<br />
Figure IV.8. The docking domain of H2A.Bbd is responsib<strong>le</strong> for anomalous remo<strong>de</strong>ling by<br />
SWI/SNF (A) RSC remo<strong>de</strong>ling reaction was performed on H2A.ddBbd nuc<strong>le</strong>osomes as <strong>de</strong>scribed in<br />
Figure IV.6. RSC activity was normalized to SWI/SNF as <strong>de</strong>scribed in the text. Lanes 1-7 represent<br />
HaeIII digestion of control H2A.ddBbd nuc<strong>le</strong>osomes (incubated in absence of ATP) at different time<br />
points. Similarly, lanes 8-14 represent HaeIII digestion of RSC remo<strong>de</strong><strong>le</strong>d nuc<strong>le</strong>osomes. HaeIII<br />
concentration was kept at 5 units/μl. Times of digestion with HaeIII and the positions of the different<br />
dyads are indicated. Free DNA, in the same condition, was digested for 1 minute (Lane 15). (B)<br />
Quantification of HaeIII accessibility of unremo<strong>de</strong><strong>le</strong>d and RSC remo<strong>de</strong><strong>le</strong>d nuc<strong>le</strong>osomes at 16 minute<br />
time point from (A). Light grey bars indicate unremo<strong>de</strong><strong>le</strong>d nuc<strong>le</strong>osomes whi<strong>le</strong> dark grey bars represent<br />
remo<strong>de</strong><strong>le</strong>d H2A.ddBbd nuc<strong>le</strong>osomes. Positions of respective dyads are <strong>de</strong>noted on x-axis. (C) Figure<br />
II.4C, lower right panel, reproduced here for com<strong>par</strong>ison of accessibility profi<strong>le</strong> of remosomes (from<br />
conventional nuc<strong>le</strong>osomes) to that of remo<strong>de</strong><strong>le</strong>d H2A.dd.Bbd nuc<strong>le</strong>osomes.<br />
IV.3 Discussion<br />
- ATP/+ RSC + ATP/+ RSC<br />
.5’ 1’ 2’ 4’ 8’ 16’ 32’ .5’ 1’ 2’ 4’ 8’ 16’ 32’ DNA<br />
1 2 3 4 5 6 7 8 9 10 1112 13 1415<br />
-D7<br />
-D6<br />
-D5<br />
-D4<br />
-D3<br />
-D2<br />
-D1<br />
-D0<br />
% c<strong>le</strong>avage<br />
% c<strong>le</strong>avage<br />
In the present work we have studied the ro<strong>le</strong> of H2A docking domain in nuc<strong>le</strong>osome<br />
mobilization mediated by SWI/SNF and RSC. Nuc<strong>le</strong>osome sliding assays using H2A C-<br />
terminal <strong>de</strong><strong>le</strong>tion as well the H2A.ddBbd chimeric proteins c<strong>le</strong>arly <strong>de</strong>monstrated the<br />
importance of H2A docking domain in this process (Figure IV.3, 4 and 5). It is important to<br />
note that neither SWI/SNF binding nor ATPase activity is affected on H2A.Bbd nuc<strong>le</strong>osomes<br />
(Angelov et al., 2004). The results presented here rather indicate towards an active structural<br />
ro<strong>le</strong> of histone octamer in chromatin remo<strong>de</strong>ling process. SWI/SNF and RSC <strong>de</strong>pen<strong>de</strong>nt<br />
remo<strong>de</strong>ling of conventional nuc<strong>le</strong>osomes starts with unwrapping and/or pumping DNA from<br />
152<br />
80<br />
60<br />
40<br />
20<br />
0<br />
C<br />
80<br />
60<br />
40<br />
20<br />
0<br />
-ATP/+RSC<br />
+ATP/+RSC<br />
D 0 D 1 D 2 D 3 D 4 D 5 D 6 D 7<br />
D 0<br />
D 1<br />
D 2<br />
D 3<br />
D 4<br />
−ATP<br />
+ATP<br />
D 5<br />
D 6<br />
D 7
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