Extended Data Figure 6: Interactions between DNMT3A and DNA.

a, Enzymatic activities of wild-type and mutant ADD–CD–C^DNMT3L. Residues on the missing loop (residues 831–846) were mutated for the in vitro DNA methyltransferase activity assay. Error bars, s.d. for triplicate experiments. Mutating above residues leads to loss of activity of ADD–CD–C^DNMT3L, supporting their important role in catalysis or DNA recognition. The missing loop in the ADD–CD–C^DNMT3L structure is equivalent to a DNA-binding loop in the HhaI–DNA structure. b, DNA has no effect on the interaction between histone H3 and DNMT3A. Left, isothermal titration calorimetry enthalpy plot for the binding of isolated ADD domain (in cell) to histone H3 peptide (residues 1–12, in syringe), with the estimated binding affinities (K_d) listed. Right, superimposed isothermal titration calorimetry enthalpy plots for the binding of ADD–CD–C^DNMT3L (in cell) to histone H3 peptide (residues 1–12, in syringe) in the absence or presence of dsDNA. The estimated binding affinities (K_d) are listed. Histone H3 peptide has comparable binding affinity to the ADD domain alone (1.75 μM) and ADD–CD–C^DNMT3L in autoinhibitory form (2.14 μM), and the addition of DNA was not able to enhance the binding affinity further. The presence of DNA led to a slight decrease in the binding affinity between histone H3 peptide and ADD–CD–C^DNMT3L, which may have resulted from slight precipitation of the protein caused by the high concentration of DNA used for titration. c, Electrophoretic mobility-shift assays for DNMT3A proteins in the absence or presence of histone H3 peptide, with protein concentrations indicated. H3–ADD–CD represents a fusion protein with histone H3 (residues 1–20) at the N terminus of ADD–CD. The assays showed that CD–C^DNMT3L strongly bound to the FAM-labelled DNA duplex, whereas the existence of the ADD domain markedly decreased DNA-binding affinity, which was partly restored by the addition of histone H3 peptide or largely restored by H3–ADD–CD fusion protein.