FIG. 3.

Swi2p motif V is required for nucleosome mobilization by SWI/SNF. (A) Schematic of the 343-bp 601 DNA fragment. Numbers at the bottom indicate base pairs. (B) A representative native PAGE analysis of a 343-bp mononucleosome incubated with WT, ΔSTRAGGLG, or R1164A SWI/SNF complexes. Samples were taken from the reactions at indicated times, incubated with excess dsDNA and glycerol to remove SWI/SNF, and electrophoresed on 4% TBE PAGE gels. (C) Exo III mapping of nucleosome positions after SWI/SNF remodeling confirms mobilization of nucleosomes on the 601 substrate. A 3 nM concentration of DNA fragment (EcoRI-PstI) labeled at the PstI site was incubated with 3 nM SWI/SNF complex (WT or ΔSTRAGGLG) and 3 mM ATP in 1× remodeling buffer. At indicated times, apyrase was added to stop remodeling, which was followed by incubation with Exo III to identify nucleosome boundaries. After deproteination, samples were examined on DNA sequencing gels. WT, wild-type SWI/SNF; no enz, no remodeling enzyme; DNA, nucleosome-free DNA. Dots indicate novel Exo III stops generated after remodeling.