FIG 5.
Acetyltransferase activity of TIP60 is required for PARP-1 dynamics on DNA damage sites. (A) Immunoblot analysis of TIP60 knockdown by shTIP60 in MEF cells in which PARP-1–GFP was stably expressed. Further reconstitution by eTIP60 or by eTIPM was monitored by immunoblot analysis. Anti-GFP and anti-PARP-1 were used to monitor PARP-1–GFP, anti-HA was used for eTIP60 or eTIPM, and anti-TIP60 was used for endogenous TIP60. Anti-beta-actin also was used. (B) FRAP analysis to monitor the dynamics of PARP-1–GFP. One side of the PARP-1–GFP accumulated at the microirradiated area was photobleached. TIP60 knockdown cells that were reconstituted with eTIP60 or eTIPM were used. Bar, 10 μm. (C) Fluorescent recovery of the GFP signal of PARP-1–GFP in either the unirradiated (left; eTIP60 in nondamaged area, 97.9% ± 5.5% [n = 15]; eTIPM, 99.3% ± 8.2% [n = 15]) or microirradiated area (right; eTIP60 in damaged area, 72.2% ± 8.7% [n = 15]; eTIPM, 45.2% ± 18.6% [n = 14]; P value of 4.4 × 10−5 between eTIP60 and eTIPM at the damaged area) was quantified and plotted.