Figure 4.
TDP1 recruitment to DNA damage sites is determined by PARP1 activity in addition to TDP1 SUMOylation. (A) Representative images showing the recruitment of wild-type TDP1 (GFP-TDP1WT) or SUMOylation mutant TDP1 (GFP-TDP1K111R) transiently expressed in HCT116 cells in response to laser-induced DNA damage. Cells expressing the ectopic proteins were kept untreated or pretreated for 2 h with the PARP inhibitor (ABT-888, 5 µM) and were analyzed by micro-irradiation with live cell microscopy and photobleaching (FRAP analysis). A subnuclear spot indicated by a circle was bleached (BLH) for 300 ms and photographed at regular intervals of 5 s thereafter. Successive images taken for ∼130 s after bleaching illustrate fluorescence return into the bleached areas. (B) Quantitation of FRAP data (n = 5) showing mean curves. Error bars represent the standard error of the mean.