Abstract
We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present electron microscopy studies revealing the dynamic nature of the complex and a 21 Å resolution reconstruction of the intact complex provides details not apparent in previously reported structures, including a large central cavity of sufficient size to accommodate a nucleosome.
Highlights
A) Top - time-course gel-based histone exchange assay
Bottom - H2A/Htz1 incorporation curves based on a scan of the gel above
The remaining particles were divided into five groups and subjected to further analysis
Summary
A) Top - time-course gel-based histone exchange assay. Bottom - H2A/Htz1 incorporation curves based on a scan of the gel above.
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