ATPase Activity Assays.

The ATPase assays were performed using the EnzChek Phosphate Assay Kit (Molecular Probes). The reactions were carried out on 1.0 µM MORC3 His-ATPase or His-ATPase–CW, in the presence and absence of 1 µM 601 DNA or 50 µM unmodified H3K4me0 or H3K4me3 peptide (1 to 12) in a buffer containing 50 mM Tris⋅HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl₂, 0.1 mM sodium azide, 200 µM MESG, and 1 U of PNP. The reaction was started by adding 2 mM ATP to the mixture at room temperature, and the release of inorganic phosphate was monitored by measuring the absorbance at 360 nm on a NanoDrop 2000c spectrophotometer (Thermo Scientific). In the presence of inorganic phosphate, produced by the hydrolysis of ATP to ADP, MESG is enzymatically converted to ribose 1-phosphate and MESG by PNP, resulting in a shift in the wavelength absorbance from 330 nm for MESG to 360 nm for the product. The rate of ATP self-hydrolysis was measured in samples prepared in parallel replacing the MORC3 protein with the buffer and then subtracted from each set of measurements. Error was calculated as the SD of at least three separate experiments.