Fig. 4. Functional assays demonstrate the importance of the Activating Helix.

a Pull-down assay using immobilized hemimethylated DNA. Three independent experiments were performed. b in vitro DNA methylation activity of DNMT1 WT (black square: mean value, white square: three independent experiment values) and F631A/F632A mutant (blacks circle: mean value, white circle: three independent experiment values) for hemimethylated DNA. The vertical axis indicates the turnover frequency of the methylation reaction of DNMT1 (15 nM) after 1 h. Michaelis–Menten curve is shown as lines. Data were presented as mean values ± SD for n = 3. c Interphase extracts depleted with either control or xDNMT1 antibodies were incubated with control buffer, purified recombinant WT xDNMT1, and F506A/F507A. The chromatin fractions were isolated at the indicated times and their bound proteins, as well as inputs, were analyzed by immunoblotting. The gel image is representative of n = 3 independent experiments. d Experimental scheme in human HCT116 colon cancer cells: the endogenous DNMT1 protein is tagged with an auxin-inducible degron (AID) tag, causing the protein to be degraded after the addition of the auxin analog IAA. In this background, rescue vectors are added that encode either WT or FF/AA versions of DNMT1. The empty vector is used as a negative control. e Western blotting shows that endogenous DNMT1 is degraded upon IAA addition, whereas exogenous WT and FF/AA DNMT1 are not degraded. The repetitive of the experiment is n = 1. f Measurement of global DNA levels by luminometric methylation assay (LUMA). Cells expressing WT DNMT1 maintain global DNA methylation when endogenous DNMT1 is degraded, whereas the FF/AA mutant does not support DNA methylation maintenance. Data represent the mean ± SD of n = 3 independent experiments. Source data of Figs. 4a–c, 4e, f are provided as a Source Data file.