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. Author manuscript; available in PMC: 2010 Jul 10.
Published in final edited form as: Mol Cell. 2009 Jul 10;35(1):58–69. doi: 10.1016/j.molcel.2009.05.013

Figure 6. Dpb4 is associated with nucleosomal DNA ~15 bp in front of the ATPase domain during template commitment.

Figure 6

(A–B) Site-specific DNA photoaffinity cross-linking was carried out under similar conditions to those in Figure 5 and Figure 6 except for photoreactive DNA. Nucleosomes were assembled with a series of photo-reactive probes spanning 17 to 67 bp from the dyad axis. DNA probes contain either a 2 (panel A) or 1 bp (panel B) gap 24/25 and 35 bp from the dyad axis, respectively, in order to determine the conformational changes of ISW2 complex during remodeling. As a control cross-linking was done at positions in the absence of gap and/or ATP for each probe position. Nucleosomes were incubated with ISW2, irradiated, and treated with DNaseI and S1 nuclease as described and the ISW2 subunits separated by 4–20% polyacrylamide SDS-PAGE. The cross-linked subunits of ISW2 complex is marked as Itc1, Isw2 and Dpb4. Bottom portion of the gel shows histone cross-linking and occasionally trace amounts of undigested probe are evident (*).