PTIP interacts with PA1. A, Coomassie Blue staining of affinity-purified PTIP-containing protein complexes. Cell extracts prepared from 293T cells stably expressing SFB-PTIP were subjected to tandem affinity purification. The final eluent was subjected to SDS-PAGE and visualized by Coomassie Blue staining. Proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis and summarized in supplemental Table 1. Lines indicate protein bands corresponding to PTIP and PA1. M, molecular mass standards. B, 293T cells were transfected with plasmids encoding SFB-tagged PTIP together with plasmids encoding Myc-tagged PA1. Immunoprecipitation (IP) reactions were performed using S-protein beads and then subjected to Western blot analyses using indicated antibodies. C, endogenous interaction between PTIP and PA1 in the absence or presence of IR. For a control, anti-PTIP or anti-PA1 immunoprecipitates were immunoblotted with indicated antibodies. D, cell cycle-independent expression of PTIP and PA1. HeLa cells were arrested overnight with 0.5 μg/ml nocodazole (Noc). Mitotic cells were “shaken off” and then released into normal media (upper). T24 cells were allowed to grow to confluency for 96 h and then trypsinized and released into fresh media (lower). Samples were taken at the indicated time points and analyzed by fluorescence-activated cell sorting and Western blotting. E and F, mapping of the corresponding regions required for PTIP·PA1 interaction. Immunoprecipitation reactions were performed using S-protein beads and then subjected to Western blot analyses using antibodies as indicated. Schematic diagrams of wild-type (WT) and deletion mutants of PTIP (E) and PA1 (F) used in this study are also included. NT, N terminus; CT, C terminus.

PTIP accumulates PA1 at DNA damage sites. A, PTIP and PA1 form foci after DNA damage. SFB-PTIP 293T stable cell lines or U2OS cells grown on coverslips were treated with 10 Gy IR, 100 μg/ml mitomycin (MMC), 25 nm camptothecin (CPT), or 2.5 μm VP16. 8 h later cells were fixed, and immunostaining was carried out using indicated antibodies. B, U2OS cells with PTIP knockdown (shPTIP#1, shPTIP#2) or transfected with control vector (shCTR) were irradiated (10 Gy), allowed to recover for 6 h, fixed, and immunostained using antibodies as indicated. Western blot analysis was performed to verify the stable knockdown of PTIP (right panel).

PTIP·PA1 localization depends on a signaling cascade involves H2AX, MDC1, and RNF8. HeLa cells or HeLa cells stably expressing HA-FLAG-PTIP were transfected with control siRNA or H2AX-, MDC1-, RNF8-, PTIP-, PA1-specific siRNAs. Cells were irradiated (10 Gy), fixed, and immunostained with anti-FLAG, anti-PA1, and pH2AX antibodies. Western blot analysis was carried out to verify the knockdown of H2AX, MDC1, and RNF8 (lower panel).

PTIP foci formation depends on γH2AX·MDC1·RNF8 but not other DNA damage checkpoint or repair proteins. A, PTIP acts downstream of H2AX·MDC1·RNF8 signaling pathway in response to DNA damage. Cells deficient for various DNA damage checkpoint or repair proteins and their respective wild-type counter-parts or reconstituted cells were infected with retrovirus expressing FLAG-tagged PTIP. Cells were then irradiated (10 Gy), and immunostaining experiments were performed using anti-FLAG and anti-γH2AX antibodies 6 h later. B, both the FHA domain and RING domain of RNF8 are required for PTIP IRIF formation. RNF8^-/- MEFs stably expressing HA-tagged PTIP were infected with retroviruses expressing SFB-RNF8, SFB-RNF8-ΔFHA, or SFB-RNF8-ΔRING mutants. Immunostaining experiments were performed using anti-γH2AX and anti-HA antibodies. C, UBC13 is required for PTIP localization at or near the sites of DNA breaks. Wild-type or UBC13^-/- MEFs were infected with retroviruses expressing SFB-PTIP. Cells were irradiated (10 Gy), fixed, and immunostained with anti-FLAG and pH2AX antibodies.

MLL3 complex may not be involved in DNA damage response. A, MLL3 and UTX do not form IRIF. U2OS cells were transfected with plasmids encoding SFB-tagged UTX. Immunostaining was carried out with anti-MLL3, anti-FLAG, and anti-γH2AX antibodies 6 h after cells exposed to IR (10 Gy). CTR, control. B, PTIP or PA1 IRIF formation occurs independently of MLL3. HeLa cells or HeLa cells stably expressing HA-FLAG-PTIP were transfected with control siRNA or MLL3 specific siRNAs (siMLL3#1 and siMLL3#2). Cells were irradiated (10 Gy), fixed, and immunostained with anti-FLAG, anti-PA1, and pH2AX antibodies. Western blot analysis was carried out to verify the knockdown of MLL3 (left panel). C, FLAG-PTIP-associated proteins isolated from HeLa S cell nuclear extracts were fractionated on Superose 6 gel filtration column (Amersham Biosciences) as described in Ref. 15. Fractions were analyzed by Western blot with indicated antibodies. Positions of protein molecular weight standards are indicated at the bottom.

Distinct C-terminal BRCT domains are involved in PTIP foci formation and PTIP/53BP1 interaction. A, PTIP foci formation requires its BRCT3 and BRCT4 domains. U2OS cells transfected with plasmids encoding SFB-tagged wild-type (WT) or deletion mutants of PTIP were exposed to 10 Gy of ionizing radiation. Cells were fixed and immunostained with anti-FLAG and anti-γH2AX antibodies. B, U2OS cells were transfected with plasmids encoding SFB-tagged wild-type (WT) or mutant PTIP. Immunostaining was carried out with anti-FLAG and anti-γH2AX antibodies 6 h after cells were exposed to IR (10 Gy). C, PTIP does not bind to ubiquitin. Ubiquitin-agarose beads were incubated lysates containing either SFB-PTIP or SFB-USP3 (positive control) for 2 h, and proteins associated with ubiquitin were subjected to Western blotting with anti-FLAG antibody. D, PTIP and 53BP1 form foci independent of each other. Wild-type, 53BP1^-/-, or PTIP^-/- MEFs were irradiated, fixed, and immunostained with antibodies as indicated. E, binding to 53BP1 requires all four C-terminal BRCT domains of PTIP. 293T cells were transfected with plasmids encoding Myc-tagged 53BP1 together with plasmids encoding wild-type or deletion mutants of SFB-tagged PTIP. Cells were irradiated and immunoprecipitation (IP) reactions were conducted using S protein beads and then subjected to Western blotting using antibodies as indicated.

PTIP is important for proper DNA damage response after ionizing radiation. A, PTIP and PA1 play a minor role in G₂/M checkpoint control after DNA damage. PTIP^+/+, or PTIP^-/-, and ATM^-/- MEFs were mock-treated or irradiated (3 Gy). Similarly, U2OS cells transfected with control siRNA (siCTR), PTIP specific siRNAs (siPTIP#1 and siPTIP#2), or PA1-specific siRNAs (siPA1#1 and siPA1#2) were also mock-treated or irradiated. Cells were harvested 1 h later, and a G₂/M checkpoint assay was carried out (see “Materials and Methods”). The percentages of cells stained with phospho-H3 antibody before and after IR treatment were obtained from three individual experiments. Error bars indicate S.D. B, PTIP and PA1 are required for cell survival after ionizing radiation. PTIP^-/- cells were reconstituted with wild-type or various PTIP deletion mutants. Cell survival after irradiation was measured by clonogenic assay according to the “Materials and Methods.” The expression of PTIP or its deletion mutants in these cells were confirmed by Western blotting using indicated antibodies (lower panel). Similarly, U2OS cells were treated with control or PA1 specific siRNAs. Cell survival after IR was measured by clonogenic assay (upper panel), and Western blot analysis was performed to verify the depletion of PA1 after siRNA transfection (right panel). WT, wild type. C, a proposed model of the DNA damage responsive pathway involving PTIP. See “Discussion” for details.