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. Author manuscript; available in PMC: 2013 Dec 14.
Published in final edited form as: Mol Cell. 2012 Oct 30;48(5):723–733. doi: 10.1016/j.molcel.2012.09.026

Figure 7. Loss of CtIP restores Ku70/80 binding and NHEJ in H2A.Z depleted cells.

Figure 7

(a) Laser striping was used to create DNA damage in U2OS cells expressing a non-specific shRNA (shCon) or shRNA targeting either H2A.Z, CtIP or both. Cells were stained for γH2AX and Ku70 15 minutes after laser striping. (b) 293T cells were transfected with non-specific shRNA (shCon), or shRNA targeting either H2A.Z or CtIP. 293T cells expressing an shRNA resistant HA-H2A.Z protein (HA-H2A.ZR) were also used. Cells were subsequently transfected with vector (Vec) or p84-ZFN (ZFN) and ChIP analysis carried out using antibody to Ku70/80 and primers located 500bp to the right of the DSB. Results ± SD (n = 3). (c) Cells stably expressing the NHEJ-GFP reporter system and either a non-specific shRNA (shCon) or shRNA targeting H2A.Z, CtIP or both were transfected with the I-Sce1 endonuclease (+). The percent of GFP positive cells is shown. Results expressed ± SD (n = 3). (d) Cells with the integrated altNHEJ-GFP reporter system were transfected with a non-specific shRNA (shCon) or shRNA targeting H2A.Z, CtIP or both. Following transfection of I-Sce1 endonuclease (+), the percent of GFP positive cells was measured. Results expressed ± SD (n = 3). (e) Model for function of H2A.Z in DSB repair. Legend: Yellow = H2A nucleosomes; Orange = H2A.Z nucleosomes. Ku = Ku70/80; Ac = acetylation; Ub = ubiquitination. See figure S7.