Dnmt1 protein domains. Full-length and truncated Dnmt1 constructs are shown with conserved domains colored as follows: DMAP1, red; RFTS domain, yellow; CXXC domain, teal; two BAH domains, blue; and methyltransferase domain, magenta.

The RFTS domain is an inhibitor of naked DNA binding. A, RFTS domain-containing Dnmt1 (amino acids 351–1616) and RFTS domain-lacking Dnmt1 (amino acids 621–1616) (0.5–10 μm) were incubated with 100 ng of 12-bp hemimethylated oligonucleotide and analyzed for DNA binding via an EMSA. DNA was detected with SYBR Green (upper panel), and protein was detected with Coomassie Blue (lower panel). The RFTS domain-lacking protein exhibited superior DNA binding to the RFTS domain-containing protein as indicated by the shift in the electrophoretic mobility of the DNA observed in lanes 4–7. B, the RFTS domain (0.5–5.0 μm) was added in trans to assays containing 5 μm RFTS domain-lacking Dnmt1 and 100 ng of hemimethylated oligonucleotide. The RFTS domain inhibited DNA binding to the RFTS domain-lacking protein, which allowed the DNA to migrate rapidly. The sequences of all DNA oligonucleotides are presented in supplemental Table 1.

Dnmt1 binding to polynucleosomes is inhibited by the RFTS domain. A, RFTS domain-containing Dnmt1 (amino acids 351–1616) and RFTS domain-lacking Dnmt1 (amino acids 621–1616) (0.5–5.0 μm) were incubated with polynucleosomes (150 ng of DNA) and analyzed for binding via an EMSA. The SYBR Green-stained gel shows that polynucleosomal DNA ran as a typical smeary ladder (lane 1). The RFTS domain-lacking protein exhibited enhanced polynucleosome-binding ability compared with the RFTS domain-containing protein as indicated by the shift in mobility of the DNA bands, which were retained in the wells upon protein interaction. B, the polynucleosome-binding ability of all protein constructs was investigated via EMSA. Protein (5 μm) was incubated with 150 ng of polynucleosomal DNA. Constructs containing the RFTS domain (lanes 3–5) showed impaired polynucleosome binding. The CXXC domain-only constructs exhibited tight binding to polynucleosomes (lanes 6 and 7). C, the RFTS domain inhibited polynucleosome binding to the CXXC domain. The RFTS domain (0.5–5 μm) was added in trans to assays containing 5 μm CXXC domain (amino acids 621–698) and 150 ng of polynucleosomes. The RFTS domain inhibited DNA binding to the CXXC domain as indicated by increased mobility of the DNA. D, the RFTS domain (0.5–5.0 μm) was added in trans to assays containing 5 μm RFTS domain-lacking protein and 150 ng of polynucleosomes. The isolated RFTS domain did not dissociate polynucleosomes from the 621–1616 form of Dnmt1.

The RFTS domain is an endogenous inhibitor of Dnmt1 activity. A, schematic of the GlaI-coupled Dnmt1 assay. B, time and Dnmt1 dependence of the GlaI-coupled assay with 20 nm DNA substrate. With the RFTS domain-lacking protein (amino acids 621–1616), robust increases in fluorescence that were dependent on the concentration of Dnmt1 were observed. C, autoinhibition of RFTS domain-containing Dnmt1. At 20 nm DNA substrate, 2 nm RFTS domain-lacking Dnmt1 construct (amino acids 621–1616) neared completion in 30 min, whereas no reaction progress was observed with 2 nm RFTS domain-containing Dnmt1 construct (amino acids 351–1616). RFU, relative fluorescence units.

The RFTS domain is a DNA-competitive inhibitor of methyltransferase activity. A, DNA substrate-dependent methyl transfer rates for the RFTS domain-lacking protein (amino acids 621–1616; ●) and RFTS domain-containing protein (amino acids 351–1616; ■) were measured at 1 mm AdoMet. The RFTS domain conferred an ∼40-fold reduction in k_cat and an ∼15-fold increase in K_m for the DNA substrate. B, the RFTS domain (amino acids 351–600) was used in trans as an inhibitor of the methyltransferase activity of the RFTS domain-lacking protein (amino acids 621–1616). The double reciprocal plot of the data with no RFTS domain (●), 100 nm RFTS domain (■), 250 nm RFTS domain (▲), and 500 nm RFTS domain (♦) clearly shows that the RFTS domain is competitive with respect to the DNA substrate. Using nonlinear regression to fit the alteration in K_m(app), a K_i of 103 ± 11 nm was determined.

Structure of the Dnmt1 RFTS domain. The RFTS domain is shown in a stereo ribbon representation with lobes color-coded: β-barrel, yellow; zinc-binding motif, orange; and α-helical bundle, green. The zinc atom is depicted as a gray sphere with interacting residues in stick format. Arrows point to disordered regions.

Two models of autoinhibition in Dnmt1. Shown are electrostatic surface representations, generated with a gradient from −10 (red) to 10 (blue) kT/e, of Dnmt1 from BAH1 to the C terminus. In A, derived from Protein Data Bank code 3PT6, a structure not including the RFTS domain, the acidic linker (magenta) between BAH1 and the CXXC (green) domains blocks productive DNA access to the active site (9). In B, consistent with our observations (Figs. 2B and 5B) that the RFTS domain competes for DNA binding with CXXC domain-containing constructs such as the 621–1616 construct, Dnmt1 is displayed with the CXXC domain and acidic linker excluded as in Protein Data Bank code 3PTA. The RFTS domain is depicted in an orientation in which three acidic loops may descend on three basic patches in the methyltransferase domain. The DNA-competitive nature of RFTS domain inhibition suggests that the RFTS domain binds to the exclusion of DNA and may be released by an RFTS-targeted Dnmt1 activator, such as Uhrf1.