Figure 5.

Chromatin remodeling and DNA damage repair factor recruitment are ATP dependent. (A) DSBs were introduced in WT HeLa cells expressing H2B-PAGFP in the presence (top) or absence (bottom) of ATP-depleting conditions of 10 mM 2-deoxyglucose and 10 mM Na-azide. Pre-UV, post-UV, and 180 s post-UV time series images are shown. Bar, 15 μm. (B) DSBs were introduced in WT MEFs expressing Nbs1-GFP (incubated with Hoechst dye) either in the presence (bottom) or absence (top) of ATP-depleting conditions (regions denoted by yellow rectangles), followed for 10 min by photobleaching of the Nbs1-GFP signal in regions covering both DNA-damaged and nondamaged chromatin (denoted by red squares) and the recovery of the Nbs1-GFP signal monitored over time (right). NBS1-GFP is not recruited to the UV laser–exposed region in the ATP-depleted cells; however, the protein exhibits rapid mobility in both the damaged and nondamaged regions in normal and ATP-depleted cells. Bar, 8 μm. (C) WT MEFs expressing GFP-53BP1 were incubated in ATP-depleting conditions, exposed to 2 Gy γ irradiation, and monitored for 20 min after irradiation before being fixed and immunolabeled against γ-H2AX. GFP-53BP1 failed to be recruited to IRIF even though a weak γ-H2AX–containing IRIF response exists. Bar, 8 μm.