Figure 4.

Effect of the PBD on protein mobility in S-phase cells. (A) Half nucleus FRAP series of C2C12 cells expressing GFP-Dnmt1^wt and GFP-Dnmt1^Q162E, PBD-GFP and GFP alone as indicated in the second column. Cell-cycle stages were identified by the subnuclear pattern of co-expressed RFP-PCNA (first column). Selected pre- and postbleach frames are shown in false color. Lower signal-to-noise ratio of FRAP series images is due to the higher imaging frame rate. Rectangles indicate the bleached ROI. The column marked with ∼t_1/2 displays frames corresponding to the half time of fluorescence recovery. The column marked with ∼t_∞ displays the frames corresponding to approximately the time points when fluorescence recovery reached the plateau. Bar: 5 µm. (B) Quantitative evaluation of FRAP experiments. Wild-type Dnmt1 shows a small but significant decrease in mobility in early/mid S-phase (dark blue curve) as compared to G1 phase (light-blue curve). A similar mobility shift between early/mid S and non-S-phase cells is also seen for PBD-GFP (dark green and green curve, respectively) and for GFP-Ligase (dark gray and gray curve, respectively). No such shift is observed for the PCNA-binding-deficient Dnmt1 mutant (red and orange curve, respectively). GFP alone (light-green curve) is shown as reference. For clarity only every fourth time point is displayed; data are represented as mean ± SEM. Kinetic shifts are indicated by shadings in the enlarged inset.