Figure 4.

SUMOylation of CtIP in S phase is dependent on cyclin-dependent kinase and ATR activities and an interaction with PCNA. (A–E, H) HeLa His₁₀-SUMO-2 cells were treated as indicated, portioned into input and His PD fractions and processed accordingly before SDS-PAGE and immunoblotting. * indicates an exogenous CtIP immunoreactive band that is not of interest; we speculate it is either lower molecular weight SUMO-2-modified CtIP or unmodified tagged-CtIP retained in the His PD fraction due to overexpression. (A) HeLa His₁₀-SUMO-2 cells were transfected with FLAG-tagged wildtype (WT) CtIP or a C-terminal truncation mutant (D6) (see Supplementary Figure S7A) or mock transfected. Shown is a representative result of four independent experiments. (B) HeLa His₁₀-SUMO-2 cells were synchronized by double thymidine block and released to mid-S phase for 1 h in plain media, then for 2.5 h in the presence of 0.1% DMSO (vehicle control), 25 μM roscovitine, 2.5 μM AZD5438, or 10 μM RO-3306. Asynchronous cells (async) were treated in 0.1% DMSO for 2.5 h. Shown is a representative result of three independent experiments. (C) HeLa His₁₀-SUMO-2 cells were synchronized to mid-S phase. 24 h prior to harvest, they were transfected with GFP-CtIP-WT or substitution mutants at residue T847, or mock transfected. Shown is a representative result of two independent experiments. (D) HeLa His₁₀-SUMO-2 cells were left asynchronous or synchronized by double thymidine block and released for 3 h to mid-S phase in the presence of 0.2% DMSO, 10 μM KU-55933 (ATM inhibitor), 20 μM ETP-46464 or 20 μM VE-821 (ATR inhibitors), or 1 μM NU-7441 (DNA-PKcs inhibitor). Shown is a representative result of at least four independent experiments. (E) HeLa His₁₀-SUMO-2 cells were cells were left asynchronous or synchronized to mid-S phase. 24 h prior to harvest, they were transfected with HA-tagged WT-CtIP or an alanine substitution mutant at residues S664, S679 and S745, or mock transfected. Shown is a representative result of two independent experiments. (F) U-2 OS cells were co-transfected with RFP-PCNA and either GFP empty vector or GFP-CtIP and processed for immunoprecipitation (IP) of GFP. Prior to IP, a portion of lysate was saved as an input control. (G) As in (F), except cells were depleted of endogenous CtIP by siRNA, then co-transfected with RFP-PCNA and either GFP empty vector, GFP-CtIP-WT or -Δ515–518. Shown is a representative result of at least six (F) or three (G) independent experiments. (H) HeLa His₁₀-SUMO-2 cells were synchronized to mid-S phase. 24 h prior to harvest, they were transfected with GFP-CtIP-WT or the Δ515–518 deletion mutant. Shown is a representative result of two independent experiments.