AbstractsBiology & Animal Science

Mechanism of ySWI/SNF and RSC Catalyzed Nucleosome Sliding

by Ning Liu

Institution: University of Rochester
Degree: PhD
Year: 2011
Keywords: Nucleosome; Nucleosome Sliding; ySWI/SNF
Record ID: 1921346
Full text PDF: http://hdl.handle.net/1802/15885


Packaging of genome DNA into nucleosomes and higher-order chromatin structures in the eukaryotic cell nucleus greatly inhibits the availability of DNA for various nuclear processes, such as transcription, replication and DNA repair. Eukaryotic cells have developed several mechanisms to modulate chromatin structures and increase the accessibility of DNA, one of which involves the activity of ATP-dependent nucleosome remodeling complexes. The multiple subunit-containing SWI/SNF complex and related RSC complex are two of the most extensively studied chromatin remodeling complexes. However, the mechanism by which these complexes uses energy derived from ATP hydrolysis to perturb histone-DNA interactions and increase accessibility of DNA in nucleosomes is not well understood. A major outcome of the activity of the SWI/SNF and RSC complexes is nucleosome mobilization or sliding, whereby the nucleosome is translocated along a stretch of DNA. Based on previous studies, there are two proposed predominant mechanisms for sliding: one involves the twist diffusion of the DNA over the core histone octamer surface like a corkscrew; a second involves dissociation of DNA at the edge of the nucleosome with re-association of DNA inside the nucleosome which forms a DNA bulge on the octamer surface that can translocate around the nucleosome. Previous work from our lab has shown that remodeling does not occur via a pure twist-diffusion mechanism (Ayoagi et al., 2002). In order to examine the bulge propagation model we synthesized a nucleosome substrate which creates a steric hindrance to the hypothetical DNA loop. I demonstrated that the H2A N-terminal or C-terminal tails of the nucleosome core can be crosslinked together by BM[PEO]4, a 17.82Ǻ bis-maleimido sufhydryl-reactive cross-linker. This forms a proteinaceous loop that is expected to inhibit the formation and/or translocation of a DNA bulge on the nucleosome surface. I found that the crosslinking does not dampen SWI/SNF and ATP-dependent increases in EcoRV restriction enzyme accessibility within the nucleosome. However, we observed that the crosslinking does restrict nucleosome movement to the end of the DNA fragment as shown by gel mobility and ExoIII digestion assays. These results suggest that these remodelers do not catalyze nucleosome sliding by a pure loop-recapture mechanism. One solution could be that the crosslinking would not block the propagation of topological stress through the nucleosome, assuming that SWI/SNF and RSC disrupt histone-DNA contacts at least partially through twist-diffusion model. Another solution could be that crosslinking inhibits the formation of large DNA loop, but remodeler uses small loops for sliding which still make DNA accessible to the restriction enzyme cutting. However I find that the loop inhibits complete translocation of nucleosome to the end of the DNA fragment, suggesting that the crosslink constrains the size of the region required for generation of torsional stress. These results provide significant new insights into the remodeling…