Xenbase Image ID: 171717

Fig. 5. CDCA7 ICF mutants are defective in chromatin recruitment of the HELLS–CDCA7 complex. (A) Alignment of CDCA7 zinc finger domains from the indicated species. The 4CXXC motif is underlined, and residues with identified ICF mutations (*) are highlighted in red. (B) Coomassie-stained gel of a pulldown of DNA beads incubated with MBP–CDCA7e harboring the indicated ICF mutations. Uncoupled beads (Bottom) were used to control for nonspecific binding. Representative of n = 2 independent experiments. (C) Western blot analyses of proteins copurified with chromatin beads recovered from interphase extracts mock depleted or depleted of CDCA7e. Beads coated with 19 × 601 naked DNA were chromatinized in interphase extract for 90 min before addition of 1 μM recombinant MBP–CDCA7e harboring the indicated ICF mutations. Following an additional 60-min incubation, chromatin beads were recovered. Representative of n = 2 independent experiments. (D) Restriction enzyme accessibility nucleosome remodeling assay with HELLS–CBP and MBP–CDCA7e with the indicated ICF mutations. The 601-positioned mononucleosomes with a 34 and 15 bp flanking DNA on the 5′ and 3′ end, respectively, incubated with the indicated remodeling proteins and MspI endonuclease. Productive nucleosome sliding exposes an MspI site, resulting in cleaved DNA (arrow). DNA was resolved on a 10% polyacrylamide gel and visualized with SYBR Gold. 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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