Figure 5.

p53 heterodimerizes with WT and R882H DNMT3A. A, increasing concentrations of DNMT3L (purple square) or p53^WT (▾) to a fixed concentration of DNMT3A^R882H led to a robust increase in anisotropy, whereas DNMT3A^WT did not display a significant change in anisotropy by the titration of DNMT3L (green circle), p53^WT (red triangle), or p53^R248W (blue diamond). B, EMSA and C, EMSA band densitometry (lanes 1 and 5) show increasing concentrations of p53^WT (lanes 3-5) to a constant concentration of DNMT3A^R882H leads to disappearance of the DNMT3A^R882H band and formation of a higher order structure (see arrows, lanes 3-5). In A, DNMT3A^WT and DNMT3A^R882H concentrations were 2.5 μm. Single point anisotropy measurements were taken after increasing concentrations of DNMT3L (green circle), p53^WT (red triangle), and p53^R248W (blue diamond) were added to DNA bound (250 nm 5′ 6-FAM-labeled GCbox30) DNMT3A^WT and DNMT3A^R882H and allowed to incubate at room temperature for 5 min. Measurements were taken using a fluorometer equipped with polarizing filters (excitation, 485 nm; emission, 520 nm). In B, gel shift assays were carried out as described in Holz-Schietinger et al. (28) and other than samples were run on native 4.5% polyacrylamide gels and binding reactions were performed at 37 °C (lane 1). For p53^WT supershifting, varying concentrations of p53^WT were preincubated for 30 min at 37 °C with DNMT3A^R882H before the addition of DNA. A and C reflect the results (mean ± S.D.) of 2 independent experiments.