FIGURE 5.
LCA is a direct inhibitor of RFTS-containing Dnmt1. A, DSF was used to determine the Tm of RFTS-containing Dnmt1 (351–1600) in the presence (●) and absence (■) of 50 μm LCA. Fitting the observed melting traces to the Boltzmann equation gives Tm values of 47.4 ± 0.1 °C and 49.3 ± 0.1 °C in the absence and presence of LCA, respectively. The nearly 2 °C shift in the observed Tm is indicative of direct binding. Fl, fluorescence. B, concentration-dependent inhibition by LCA was investigated at 100 nm oligonucleotide 8006 and 100 μm AdoMet using 10 nm RFTS-containing Dnmt1 (amino acids 351–1600). The percent activity of each condition was determined by comparison to an uninhibited DMSO-containing control. Fitting the data using a Hill slope of −1 gives an IC50 of 19.1 ± 1.7 μm.