Figure 4: Hm-DNA impairs the intramolecular interaction of UHRF1 and facilitates its histone recognition.
From: Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition
(a) Hm-DNA impairs the intramolecular interaction of PHD–SRA. The SRA was incubated with GST-tagged PHD in the presence of increasing concentrations of hm-DNA and immobilized on glutathione resin. The bound proteins were analysed in SDS–PAGE and Coomassie blue staining (left) and quantified by band densitometry (right). Error bars, s.d. for triplicate experiments. (b) Purified fragments of UHRF1 were analysed by histone peptide (H3K9me0) pull-down assay as described in Fig. 1b. (c) Hm-DNA impairs the intramolecular interaction of TTD–Spacer. SRA–Spacer was incubated with GST-tagged TTD–PHD or TTD in the presence of increasing concentrations of hm-DNA and analysed in pull-down experiment as described in a. The quantified band densitometries are indicated below the Coomassie blue staining. Error bars, s.d. for triplicate experiments. (d) Histone peptide pull-down assay using UHRF1 mutants as indicated. The assays were performed in the presence (+DTT) or absence (−DTT) of 15 mM DTT.
