Hydroxyurea (HU)-mediated DNA damage induces SET7-dependent methylation of TIP60 and induces homologous recombination (HR). (A) HCT116 cells were treated with 5 mM HU for 4 h. IPs using an anti-methyl lysine antibody were performed. Methyl lysine levels were normalized by input of TIP60. (B) HCT116 cells were arrested at the G1/S checkpoint by double thymidine block/release, and the cells were then treated with 5 mM HU for 4 h. IPs using an anti-methyl lysine antibody were performed. Methyl lysine levels were normalized by input of TIP60. Results were shown as the mean ± SEM; n = 3, ***P < 0.001, **P < 0.01. (C) Schematic diagram of the HR reporter. PcDNA3.1-SET7, Flag-TIP60 WT, or Flag-TIP60 K137R was subjected to HR assay. Results were shown as the mean ± SEM; n = 5, *P < 0.05, N.S: no significant difference. (D) HCT116 cells with TIP60 knockdown were transfected with TIP60 WT or TIP60 K137R. RPA foci was examined following 5 mM HU treatment for 4 h. (E) HCT116 shTIP60 cells overexpressing SET7 were transfected with the indicated plasmid. Then, cells were treated with HU and subjected to a neutral comet assay. The tail moment was determined using CaspLab software, and 90 individual comets were counted for each sample (lower panel). Results were shown as the mean ± SEM; n = 90, ***P < 0.001.
