Phosphorylation-dependent formation of BRCA1/CtIP complex is cell cycle regulated. (A and B) HeLa cells were arrested in M phase by treatment with nocodazole (1 μg/ml) for 24 h (A) or at G₁/S boundary by double thymidine block (B). Cells were then released and harvested at the indicated time points. Immunoprecipitations and immunoblotting were performed as indicated. Since Chk2 protein level is not cell cycle regulated, an anti-Chk2 immunoblot was included as a protein loading control. Both anti-cyclin B and anti-phospho-histone H3 blots were used as controls for cell cycle progress. Cell cycle distributions were analyzed by fluorescence-activated cell sorting and summarized here.

CtIP is required for Chk1 activation after DNA damage. (A) HCC1937 cells or HCC1937-BRCA1 cells were treated with or without irradiation (IR; 8 Gy). Cell lysates were collected and immunoblotted with anti-phospho-Chk1 (anti-pS317) or anti-Chk1 antibodies. (B) HeLa cells were transfected with control, CtIP or BACH1 siRNAs. Cells were treated with or without irradiation (IR; 8 Gy). Cell lysates were collected and immunoblotted with indicated antibodies. (C) HeLa cells were transfected with CtIP siRNA after infection with lentivirus expressing wild type (WT) or the S327A mutant of CtIP. Cells were treated with irradiation (IR; 8 Gy) at 48 h after CtIP siRNA transfection. Cell lysates were immunoblotted with anti-phospho-Chk1 (anti-pS317) or anti-Chk1 antibodies.

The BRCA1 BRCT domain interacts specifically with phosphorylated CtIP. (A) Flag-tagged CtIP deletion mutants were expressed in 293T cells. Cell lysates were incubated with GST-BRCA1-BRCT protein immobilized on beads. Associated CtIP proteins were eluted and detected with anti-Flag antibody (upper panels). Whole-cell lysates were also blotted with anti-Flag antibody to ensure similar expression levels of various CtIP mutants (lower panels). (B) Potential phospho-motif on CtIP. (C to E) Wild-type or S327A mutant of CtIP was expressed in 293T cells. Cell lysates were incubated with beads containing GST-BRCA1-BRCT for pull-down assay (C) or incubated with the indicated antibodies for immunoprecipitation (D and E). Associated or immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted with indicated antibodies. (F) Endogenous CtIP from 293T cell lysate was immunoprecipitated with anti-CtIP antibody, followed by treatment with or without λ phosphatase (PPase) or phosphatase inhibitor. Immunoprecipitated CtIP was then immunoblotted with anti-phospho-S327 antibody (upper panels). An identical blot was also blotted with anti-CtIP antibody to ensure similar amounts of CtIP in these experiments. (G) Phosphorylated CtIP peptide competes with endogenous CtIP for BRCA1 binding. 293T cell lysates were incubated with beads containing GST-BRCA1-BRCT proteins in the presence of 10 μM phosphorylated peptide (pS, TRVSpSPVFGAT) or unphosphorylated control peptide (S, TRVSSPVFGAT). Endogenous CtIP associated with BRCA1 BRCT domains was eluted and detected by using anti-CtIP antibody.

Phosphorylation-dependent BRCA1 complexes participate in different cell cycle checkpoints after DNA damage. (A and B) Two checkpoint defects in HCC1937 cells. Checkpoint assays were performed as described in Materials and Methods. The percentage of phospho-histone H3 positive staining cells was measured. (C) HeLa cells were transfected with control, CtIP, BRCA1, or BACH1 siRNA. Cell lysates were immunoblotted with indicated antibodies. Irrelevant siRNA was included as a negative control. G₂ accumulation assays were performed 48 h after the initial siRNA transfection. (D) HeLa cells were transfected with control, BRCA1, BACH1, or CtIP siRNA. Transient G₂/M transition assays were carried out 48 h after the initial siRNA transfection. (E) siRNA-resistant silent mutant of CtIP (WT) and S327A mutant were expressed in 293T cells by using a lentivirus expression system. Cell lysates were immunoprecipitated with anti-Flag or anti-CtIP antibodies and blotted with anti-CtIP antibody. (F) HeLa cells were transfected with CtIP siRNA after infection with wild type (WT) or S327A lentivirus. Cell lysates were immunoblotted with anti-CtIP or anti-BRCA1 antibodies. Whole-cell lysates (WCL) of untreated cells were included as control. Transient G₂/M transition checkpoint assays were performed 48 h after CtIP siRNA transfection.