Figure 6.

SIAH2 mediates CtIP ubiquitination at K62, K78, K115, K132 and K133, which is important for resection and HR. (A) DR-GFP U2OS cells expressing either Flag-CtIP WT or Flag-CtIP-5KR were transfected with an I-SceI expression plasmid for up to 8 h. ChIP experiments were performed using an anti-Flag antibody. Results are shown as the mean ± SD (n = 3), **P < 0.01, **P < 0.01, two-tailed Student's t-test. (B) Control HeLa cells or CtIP-depleted HeLa cells reconstituted with Flag-Mock, Flag-CtIP WT or Flag-CtIP-5KR were treated with 5 Gy IR for 3 h. Chromatin fractions were subjected to western blotting with the indicated antibodies. (C) Quantification of ssDNA generated by 5’ end resection at three AsiSI-induced DSBs in either control or CtIP-depleted AsiSI-ER-U2OS cells reconstituted with Flag-Mock, Flag-CtIP WT or Flag-CtIP-5KR. Results are shown as the mean ± SD (n = 3), **P < 0.01. ns, nonsignificant, two-tailed Student's t-test. (D and E) Control HeLa cells or CtIP-depleted HeLa cells reconstituted with Flag-CtIP WT or Flag-CtIP-5KR were exposed to 5 Gy IR, fixed after 3 h, and immunostained with anti-RPA and anti-Flag antibodies (D) or anti-RAD51 and anti-Flag antibodies (E). DNA was stained with DAPI. Representative images and the percentage of cells containing >10 foci are shown. Results are shown as the mean ± SD (n = 3), **P < 0.01. ns, nonsignificant, two-tailed Student's t-test. (F) The efficiency of HR repair in either control or CtIP-depleted DR-GFP-U2OS cells reconstituted with Flag-Mock, Flag-CtIP WT or Flag-CtIP-5KR. Results are shown as the mean ± SD (n = 3), **P < 0.01. ns, nonsignificant, two-tailed Student's t-test. (G) IR-induced chromosomal aberrations were measured in the same cells described in (B) 24 h after treatment with 2 Gy of IR. Dot plot shows the number of chromosomal aberrations per cells. P values were calculated by the Mann–Whitney test. ns, nonsignificant.