Figure 3. DNA gaps block SWR1 activity when positioned 17–22 bp on either side from dyad.
All nucleosomes have a 20 bp linker DNA at both ends, and a two-nucleotide gap introduced at indicated positions. EMSA (6% native PAGE) shows the H2A.Z replacement reaction, terminated at the indicated times, using fluorescently labeled nucleosomes (4 nM), SWR1 (2 nM), and H2A.Z-3F-H2B dimer (10 nM). Nucleosome products with 0, 1, and 2 H2A.Z-3FLAG molecules are resolved (AA, AZ, ZZ). (A) Mapping of gap sites that block SWR1 activity. Left: Design of WT and gap nucleosomes. Right: (+) and (−) denote presence and absence of the AZ or ZZ species. (B) Fine mapping of the gap-sensitive region near two turns from nucleosome dyad. (C) Gaps within the sensitive region on both sides of nucleosome completely block SWR1 activity.
Figure 3—figure supplement 1. SWR1 binding to nucleosome core particle with gaps on both sides of dyad.
Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles.
Figure 3—figure supplement 2. Nucleosomal histone and DNA elements critical for SWR1 activity and model for SWR1-mediated H2A-H2B displacement.
(A) Yeast nucleosome structure PDB 1ID3 was modeled to show one face of the nucleosome and the histone-fold elements that are critical for SWR1 activation. The SWR1 footprint is shown in blue. The gap-sensitive region, 17–22 nt from dyad, is shown in cyan. Residues of H2A that affect SWR1 activity are shown in magenta. (B) Nucleosome model showing histone-DNA and histone–histone interactions that hold H2A-H2B within the nucleosome. Also shown is the gap-sensitive region, where SWR1 interacts with nucleosome DNA leading to eviction of H2A/H2B and concomitant deposition of H2A.Z/H2B.