FIGURE 2.

DNA 5-mC demethylation activities of mammalian DNMTs. The 5-mC-containing DNA substrates were subjected to incubation in 293T nuclear extracts in buffer B with 10 mm CaCl₂ and exogenously expressed EGFP (lanes 1 and 2) and mouse DNMT1 (lanes 3 and 4), DNMT3A (lanes 5 and 6), and DNMT3B (lanes 7 and 8), as well as their catalytic mutants (site-directed mutants of the DNA C-5 methylation catalytic sites of the three enzymes): DNMT1-PSC, DNMT3A-PS, and DNMT3B-PS (lanes 9–14). The extent of conversion of 5-mC to C was analyzed by the hydrolysis-TLC assay and is shown quantitatively in the histogram. The amounts of the exogenous wild-type enzyme in lanes 4, 6, and 8 were similar to those of the mutant enzymes in lanes 10, 12, and 14, respectively (Western blotting data not shown). M, mock control without incubation; R, with incubation. Error bars indicate S.D. *, p < 0.05 by t test comparing bars 4, 6, and 8 with bar 2.