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Fig. S5. Characterization of HELLS–CDCA7e ATPase and remodeling activity. (A) Entire gel from Fig. 4B. (B) Native gel nucleosome remodeling assay. Endpositioned (Left) or center-positioned mononucleosomes (Right) were incubated with the indicated remodeling proteins. Reactions were stopped, resolved on a 5% polyacrylamide gel, and visualized with SYBR Gold. Representative of n = 2 independent experiments. (C) Restriction enzyme accessibility nucleosome remodeling assay. The 601-positioned mononucleosomes with a HaeIII site 11 bp into the nucleosome with 60 bp flanking DNA on the 3′ end were incubated with the indicated remodeling proteins and HaeIII. Productive nucleosome sliding exposes the HaeIII site, resulting in cleaved DNA (arrow). DNA was resolved on a 10% polyacrylamide gel and visualized with SYBR Gold. (D and E) Quantification of ATPase activity. The indicated proteins were incubated with gamma33P ATP for the indicated times at 37 °C (D) or 16 °C (E) for the indicated time. Reaction was separated by TLC, exposed to a PhosphorStorage screen, and the fraction of hydrolyzed ATP was quantified. In E, mean and SD from n = 3 distinct replicates are displayed. Image published in: Jenness C et al. (2018) Copyright © 2018. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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