FIG 5.

H2AX K5 acetylation by TIP60 is required for efficient GFP-NBS1 turnover on damaged chromatin. (A and B) Schematics of FRAP followed by microirradiation for the undamaged area (A) and for the microirradiated area (B). (C) Expression of NBS1-GFP in U2OS cells expressing TIP60 or TIPM from the pCAGGS vector. Results for control cells expressing neither TIP60 nor NBS1-GFP are in the Empty vector lane. (D) Fluorescence recovery curves of NBS1-GFP after bleaching an unirradiated area (left; n = 12 for TIP60 and n = 13 for TIPM) or UVA-microirradiated area (right; n = 14 for TIP60 and n = 12 for TIPM). U2OS cells stably expressing TIP60 or TIPM were used. (E) Mean residence times from the analysis whose results are shown in panel D. (F) Expression of NBS1-GFP in U2OS cells expressing H2AX WT, K5R, or S139A from pCAGGS. Results for control cells expressing neither H2AXs nor NBS-GFP are in the Empty vector lane. (G) Fluorescence recovery curves of NBS1-GFP after bleaching an unirradiated area (left; n = 12 for WT, n = 10 for K5R, and n = 11 for S139A) or a UVA-microirradiated area (right; n = 12 for WT, n = 10 for K5R, and n = 10 for S139A). (H) Mean residence times from the analysis whose results are shown in panel G. Mean residence times were calculated as previously reported (28) and statistically analyzed by using the Student t test. P values are indicated. Graphs show standard deviations.