PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Antibodies and Other Materials

Rabbit anti-mouse PTIP, RNF8, RAP80, and BRCA1 polyclonal antibodies were raised against GST-PTIP (amino acids 1725–1969), GST-RNF8 (amino acids 1–324), GST-RAP80 (amino acids 1–354), and GST-BRCA1 (amino acids 1445–1812) fusion proteins, respectively. Anti-53BP1, anti-phospho-H2AX, and anti-phospho-CHK2 (T68) antibodies were previously described (32). Anti-ATM, anti-phospho-ATM (S1981), anti-phospho-p53 (S15), anti-SMC1, and anti-phospho-SMC1 (S957) were purchase from Cell Signaling Technology. Anti-H4 and anti-β-actin antibodies were purchased from Upstate and Sigma, respectively.

The siRNA duplexes were purchased from Dharmacon Research (Lafayette, CO). The siRNA sequence targeting PTIP and 53BP1 are 5′-AAG GAA GAA GAG GAA GAG GAA-3′ and 5′-AAG AUA CUC CUU GCC UGA UAA-3′, respectively. siRNAs were transfected into the cells using oligofectamine (Invitrogen) according to the manufacturer's instructions.

Cell Culture and Treatment with Ionizing Radiation

U2OS cells were cultured in RPMI 1640 medium with 10% fetal bovine serum. H2AX-, MDC1-, RNF8-, and 53BP1-deficient MEFs have been described elsewhere (32–35). The ptip^flox/− MEFs were previously described (31). RAP80-deficient MEFs were provided by Dr. Junjie Chen. All of the MEFs were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The ptip^{flox/− MEFs} were infected with Cre-expressing adenovirus (ad-Cre, University of Michigan Vector Core). For IR, the cells were irradiated by JL Spepherd ¹³⁷Cs radiation source with indicated doses. The cells were then maintained in the culture conditions for indicated time points specified in the figure legends.

Cell Lysis and Western Blot

The cells were lysed with buffer containing 0.5% Nonidet P-40, 50 mm Tris-HCl, pH 8.0, 2 mm EDTA, and 300 mm NaCl for further Western bolt analysis. For soluble and insoluble (chromatin) fractions, the cells were lysed with buffer containing 0.5% Nonidet P-40, 50 mm Tris-HCl, pH 8.0, 2 mm EDTA, and 100 mm NaCl, and insoluble pellets were resuspended in 0.2 m HCl and then were neutralized with 1 m Tris-HCl, pH 8.5.

Immunofluorescence Staining

To visualize IR-induced foci (IRIF), the cells were cultured on coverslips and treated with 5 Gy of IR followed by recovery for 4 h and then were fixed in 3% paraformaldehyde for 20 min and permeabilized in 0.5% Triton X-100 in phosphate-buffered saline for 5 min at room temperature. The samples were blocked with 5% goat serum and then incubated with primary antibody for 60 min. The samples were washed three times and incubated with secondary antibody for 30 min. The coverslips were mounted onto glass slides and visualized by a fluorescence microscope.

In Vitro Kinase Assay

Wild type, PTIP, and 53BP1-deficient cells were treated with 10 Gy of IR and allowed to recover for 1 h. The cells were lysed in NTN 300 buffer (0.5% Nonidet P-40, 50 mm Tris-HCl, pH 8.0, and 300 mm NaCl). The extract was incubated with ATM antibody and protein G beads at 4 °C for 2 h. After washing three times with the kinase buffer (10 mm HEPES, pH 7.5, 50 mm glycerophosphate, 50 mm NaCl, 10 mm MgCl, 10 mm MnCl, and 1 mm dithiothreitol), the beads were resuspended in the kinase buffer with or without ATP and incubated with purified GST-CHK2 (amino acids 1–250) fusion protein at 37 °C for 30 min. The proteins were eluted with SDS loading buffer, separated by SDS-PAGE, and analyzed by Western blot using anti-phospho-CHK2-T68 antibody.

Neutral Comet Assays

Single-cell gel electrophoretic comet assays were performed under neutral conditions (36). Briefly, ptip^flox/− MEFs with or without treatment of Ad-Cre were arrested at G₁ by treated with 2 mm thymidine for 24 h. The cell cycle profile was confirmed by flow cytometry (data not shown). The cells were irradiated with 25 Gy and incubated in culture medium at 37 °C for the indicated times. For cellular lysis, the slides were immersed in neutral lysis solution (2% sarkosyl, 0.5 m EDTA, 0.5 mg/ml proteinase K, pH 8.0) overnight at 37 °C. On the second day, after electrophoresis at 15 V for 25 min (0.6 V/cm), the slides were stained for 20 min with 10 μg/ml propidium iodide and viewed in a fluorescence microscope. The comet tail moment was analyzed by CometScore software.

Pulsed Field Gel Electrophoresis

MEFs were exposed to 80-Gy IR. At the indicated time after IR, the cells were harvested by trypsinization, washed, and resuspended in phosphate-buffered saline. Low melting point agarose was added to a final concentration of 0.8%, and 0.1-ml plugs were cast containing ∼300,000 cells of each. The plugs were incubated in a 0.5 m EDTA solution containing 2% sarkosyl and 10 μg/ml proteinase K at 56 °C for 24 h, followed by wash plugs twice with TE buffer (pH 8.0) for 2 h. Pulsed field gel electrophoresis was performed on a Bio-Rad Chef II Mapper for 36 h optimized to the 1–4-Mb range. The gels were stained with Sybr green I (Invitrogen) overnight in the dark at 4 °C and then were scanned by using a phosphorimaging device in fluorescence mode (GE HealthCare). The DNA fragmentation was detected as DNA migrated into the gel and calculated as a ratio of DNA migrated in each lane of the gel relative to the total DNA loaded (separated and nonseparated DNA). The DNA double strand breaks level was calculated as a ratio of the amount of DNA fragmentation at each recovery time point to that at time 0 following DNA damage. The mean value at each time point is from three independent experiments.

Radioresistant DNA Synthesis Assay

ptip^flox/− MEFs were treated with or without adeno-cre and plated at 40% confluency in 60-mm dishes in fresh medium containing 0.01 μCi ml⁻¹ of ¹⁴C-labeled thymidine (Amersham Biosciences). After 24 h, the medium was replaced with thymidine-free medium, and the cells were treated with 5-Gy IR in a GammaCell ¹³²Cesium source. The cells were returned to the incubator for 30 min before the addition of 20 μCi/ml of ³H-labeled thymidine (Amersham Biosciences). At the indicated time points, the cells were washed, trypsinized, pelleted, and frozen on dry ice until all of the samples were collected. The samples were lysed in 500 μl of 0.1 n NaOH, 1% SDS, 1 mm EDTA. After incubation at 65 °C for 30 min, an equal volume of 20% trichloroacetic acid was added to the samples, which were immediately and thoroughly mixed. Samples were applied to Whatman GF/A filters on a vacuum apparatus and were washed extensively with 10% trichloroacetic acid and then with 100% ethanol. After air-drying, the filters were placed in vials with 3 ml of scintillation fluid and counted on a Packard Tri-Carb 2000 liquid scintillation analyzer using the standard dual label procedure. The data are presented as the percentage of DNA synthesis at the various time points, where the percentage of DNA synthesis = (³H dpm/¹⁴C dpm) treated/(³H dpm/¹⁴C dpm) untreated × 100.

PTIP Is Downstream of H2AX, MDC1, and RNF8 following DNA DSBs

Following DNA damage, PTIP is quickly accumulated at DSBs and colocalizes with γH2AX (16–18). However, it remains unclear how this dynamic process is regulated. Following DNA damage, γH2AX recruits MDC1, then RNF8, to DSBs (24–27, 29, 37–39). This pathway regulates several downstream proteins relocation to the DNA damage sites, such as 53BP1 and the RAP80·BRCA1 complex (37–40). To study the role of PTIP in this DNA damage response pathway, we have examined IRIF of PTIP in several DNA damage response-deficient cells. Wild type, H2AX, MDC1, RNF8, 53BP1, and RAP80-deficient MEFs were treated with 5 Gy of IR. As shown in Fig. 1, PTIP failed to relocate to DSBs in the absence of H2AX, MDC1, or RNF8. But IRIF of PTIP did not require 53BP1 and RAP80, both of which are downstream of RNF8. These results suggest that the γH2AX-MDC1-RNF8 pathway controls PTIP IRIF.

PTIP Regulates 53BP1 Foci Formation following DNA Damage

Because PTIP foci formation is independent of 53BP1 and RAP80, we next examine whether PTIP is upstream of 53BP1 and the RAP80-BRCA1 complex following DNA damage. PTIP-deficient MEFs were generated from ptip^flox/− MEFs by introducing Cre recombinase (31). We confirmed that PTIP was not detected by Western blot in the PTIP-deficient MEFs (Fig. 2A). Moreover, the IRIF of RNF8, RAP80, and BRCA1 was intact in the PTIP-deficient MEFs (Fig. 2B). However, 53BP1 foci formation was abrogated in the absence of PTIP (Fig. 2B), indicating that PTIP regulates IRIF of 53BP1 but not that of the RAP80-BRCA1 complex.

PTIP and 53BP1 Regulate Phospho-ATM Association with Chromatin

It has been shown that MDC1, the upstream regulator of PTIP and 53BP1, modulated ATM association with chromatin (34), which prompted us to examine the role of PTIP and 53BP1 in the ATM association with chromatin. Following DNA damage, chromatin-associated ATM are dramatically increased (34). We also confirmed this phenomenon in the normal MEFs following DNA damage (data not shown). However, compared with that in the normal MEFs, chromatin-bound phospho-ATM and ATM were significantly reduced in the PTIP- and 53BP1-deficient MEFs (Fig. 3A). These results suggest that PTIP and 53BP1 regulate the phospho-ATM association with chromatin.

We noticed that although chromatin-associated phospho-ATM are dramatically decreased in PTIP- and 53BP1-deficient cells, the phospho-ATM in soluble fraction are significantly increased, suggesting that PTIP and 53BP1 may not affect the phosphorylation and kinase activity of ATM following DNA damage. This hypothesis was confirmed by a Western blot to detect the phospho-ATM. As shown in Fig. 3B, compared with that in the normal MEFs, ATM was still phosphorylated in the PTIP- and 53BP1-deficient MEFs following IR, although the phosphorylation level of ATM was modestly reduced. Moreover, the kinase activity of ATM from the PTIP- and 53BP1-deficient MEFs was not significantly altered in an in vitro kinase assay using recombinant CHK2 as the substrate (Fig. 3C). These results together suggest that PTIP and 53BP1 regulate the phospho-ATM and ATM association with chromatin, although they cannot affect the phosphorylation and kinase activity of ATM.

PTIP and 53BP1 Regulate ATM-dependent SMC1 Phosphorylation at the DNA Damage Sites

Because PTIP and 53BP1 regulate chromatin-bound ATM, we hypothesize that ATM substrates on the chromatin may also be regulated by PTIP and 53BP1. SMC1 is a subunit of sister chromatin cohesion complex that is incorporated into the chromatin during S phase and maintains the association between the sister chromatids (41, 42). Following DNA damage, chromatin-associated SMC1 is phosphorylated by ATM at Ser⁹⁵⁷ and Ser⁹⁶⁶ (43–45). Phosphorylated SMC1 is important for DSBs repair (43–47). To examine the role of PTIP and 53BP1 in ATM-dependent SMC1 phosphorylation, we used siRNA to down-regulate PTIP and 53BP1 in U2OS cells and found that the phosphorylation of SMC1 was abolished in the PTIP and 53BP1 depleting cells following IR (Fig. 4A), suggesting that PTIP and 53BP1 regulate the ATM-dependent SMC1 phosphorylation. Again, in the PTIP and 53BP1 depleting cells, ATM phosphorylation was not significantly altered (Fig. 4A), which is consistent with our observation in the PTIP- and 53BP1-deficient MEFs (Fig. 3B). In addition, we also examined the phosphorylation level of p53 and Chk2, another two known substrates of ATM, and found that Ser¹⁵ phosphorylation of p53 and Thr⁶⁸ phosphorylation of Chk2 were only modestly reduced in PTIP- and 53BP1-depleting cells (supplemental Fig. S1), indicating that other phosphoinositide 3-kinases including ATR and DNAPK may function redundantly with ATM to phosphorylate downstream substrates such as Chk2 and p53 following DNA damage.

Next, we examined the IRIF of phospho-SMC1 in the presence or absence of PTIP and 53BP1. As shown in Fig. 4B, phospho-SMC1 formed nuclear foci and colocalized with γH2AX and 53BP1 in control siRNA-treated cells. In contrast, the IRIF of phospho-SMC1 was abolished in the PTIP- and 53BP1-depleting cells. Furthermore, we could not detect phospho-SMC1 signals by immunostaining (Fig. 4B). In addition, the IRIF of 53BP1 was also abolished in the PTIP depleted cells, further confirming that 53BP1 foci formation is dependent on PTIP. Although the Ser⁹⁵⁷ of SMC1 is conserved from human to mouse, the +4 Asp in human has exchanges to Glu in mouse. Thus, we failed to use this anti-human phospho-SMC1 to detect mouse SMC1 by immunostaining. Nevertheless, by using siRNA to knockdown PTIP and 53BP1, we demonstrate that PTIP and 53BP1 regulate the phosphorylation of ATM substrate on the chromatin possibly because of control phospho-ATM accumulation at the DNA damage sites.

PTIP Participates in DNA Damage Repair and Intra-S Phase Checkpoint

53BP1 participates in nonhomologous end joining (NHEJ) following DNA DSBs and other types of NHEJ under physiological condition, such as immunoglobulin class switching recombination during B cell development (48–50). Because PTIP participates in the DNA damage response and recruits 53BP1 to DSBs, we wonder whether PTIP is involved in NHEJ repair. We measured the efficiency of NHEJ in the PTIP-deficient MEFs by using the comet assay under the neutral condition. As shown in Fig. 5A, the average length of the comet tail of PTIP-deficient MEFs was much longer than that in control MEFs in a time course following IR. We also performed a pulsed field gel electrophoresis-based DSB assay after IR to directly measure DNA repair efficiency (51). The fragmentation of chromosomes can be detected as DNA entering the gel and is quantized as a ratio of DNA within the lane relative to the total DNA loaded. We observed that PTIP ± cells displayed progressively less DNA damage in a time course from 0 to 18 h after IR, whereas PTIP^−/− cells displayed a moderate defect in DNA repair (Fig. 5B). These results suggest that PTIP is involved in NHEJ follow DNA double strand breaks.

Both ATM-deficient cells and cells transiently expressing SMC1 S957A mutant display defects in the intra-S phase checkpoint following DNA damage (44, 45, 52). We have tested whether PTIP participates in the S phase checkpoint in response to IR. Similar to that in the 53BP1-deficient cells (32) and SMC1 mutant cells (44), DNA damage-induced intra-S phase checkpoint activation was slightly impaired in the PTIP-deficient cells, suggesting that PTIP may play a subtle role in intra-S phase regulation.

Supplemental Data