| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Biochemistry, and 2 Medical Research Institute, Kanazawa Medical University, Daigaku 1-1, Uchinada, Kahoku-gun, Ishikawa 920-0293, Japan
3 Department of Molecular Pathology, Cancer Research Institute, Faculty of Medicine, Kanazawa University, Kanazawa, Ishikawa, 920-0934, Japan
4 Department of Environmental Sciences, Faculty of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan
5 Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Totsuka-ku, Yokohama 244-0813, Japan
6 Department of Immunology and Molecular Genetics, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
-H2AX foci, a marker of DNA DSBs, was prolonged in 53BP1-deficient cells. Thus, the elevated X-ray sensitivity in G1 phase cells was attributable to repair defect for IR-induced DNA-damage. Epistasis analysis revealed that 53BP1 plays a role in a pathway distinct from the Ku-dependent and Artemis-dependent NHEJ pathways, but requires DNA ligase IV. Strikingly, disruption of the 53BP1 gene together with inhibition of phosphatidylinositol 3-kinase family by wortmannin completely abolished colony formation by cells irradiated during G1 phase. These results demonstrate that the 53BP1-dependent repair pathway is important for survival of cells irradiated with IR during the G1 phase of the cell cycle. |
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Upon exposure to IR, p53-binding protein 1 (53BP1) (Iwabuchi et al. 1994, 1998) is rapidly redistributed to sites of DSBs and is hyperphosphorylated in an ataxia telangiectasia mutated protein (ATM)-dependent manner (Schultz et al. 2000; Xia et al. 2000; Anderson et al. 2001; Rappold et al. 2001). ATM, a protein defective in the heritable disorder ataxia telangiectasia, is a central signaling kinase in the response to DSBs (Shiloh 2003). Studies using 53BP1–/– mouse embryonic fibroblasts or small interfering RNA to inhibit 53BP1 expression have revealed that 53BP1 is required for the accumulation of p53 protein, G2/M checkpoint arrest, the intra-S-phase checkpoint in response to IR damage (DiTullio et al. 2002; Fernandez-Capetillo et al. 2002; Wang et al. 2002), and IR-stimulated phosphorylation of at least a subset of ATM substrates, including Chk2, BRCA1 and SMC1 (DiTullio et al. 2002; Wang et al. 2002). The checkpoint defects observed in 53BP1-deficient cells are modest and restricted to responses to low doses of IR, suggesting that 53BP1 has other functions in the response to IR-induced DNA damage.
DSBs are repaired by two major pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) (Jeggo 1998; Jackson 2002). HR primarily uses the undamaged sister chromatid as a DNA template allowing for accurate repair of the lesions (Johnson & Jasin 2000), and functions in late S-G2 phase (Rothkamm et al. 2003). NHEJ is an error-prone joining of DNA ends with the use of little or no sequence homology. NHEJ requires the DNA ligase IV (Lig IV)/Xrcc4 complex and DNA-dependent protein kinase (DNA-PK), which consists of the DNA end-binding subunits of Ku (Ku70 and Ku80) and the catalytic subunit (DNA-PKcs). In addition to participating in the rejoining of DSBs during V(D)J recombination, NHEJ plays a major role in the repair of IR-induced DSBs, especially during the G1 phase of the cell cycle when sister chromatids are not available (Rothkamm et al. 2003). It is thought that broken DNA termini are recognized by the Ku heterodimer, which then recruits DNA-PKcs, activating its kinase activity. This large complex protects the DNA ends from nuclease attack while facilitating the recruitment of the Lig IV/Xrcc4 heterodimer (Lieber et al. 2003; Downs & Jackson 2004). Cells lacking any of these factors show pronounced IR sensitivity and have an impaired ability to rejoin IR-induced DNA DSBs (Lobrich & Jeggo 2005).
Artemis (Moshous et al. 2001), a protein with nuclease activity, forms a physical complex with DNA-PKcs. The endonuclease activity of Artemis is required for processing the hairpin intermediate generated during V(D)J recombination (Ma et al. 2002), and it also appears to function similarly in end-processing during IR-induced DNA DSB repair (Riballo et al. 2004). Riballo et al. (2004) identified Artemis as a downstream component of ATM-dependent signaling in DSB repair. They also proposed a model for the repair of IR-induced DSBs during the G1 phase in mammalian cells, in which the majority of DSBs are rejoined by NHEJ. This process requires what they refer to as the "core NHEJ," which is composed of Lig IV/Xrcc4, Ku70/Ku80, and DNA-PKcs, wherein DNA-PKcs plays a nonessential but facilitating role. In addition, a subfraction of DSBs that require end-processing by Artemis is repaired by ATM/Artemis-dependent NHEJ. This ATM/Artemis-dependent repair pathway also requires proteins locating to sites of DSBs, including 53BP1.
We showed in a previous study that the ligation reaction catalyzed by recombinant Lig IV/Xrcc4 is enhanced by a purified polypeptide corresponding to the domain of 53BP1 responsible for focus formation (Iwabuchi et al. 2003). This suggests that 53BP1 participates in a DNA damage repair pathway. To investigate the role of 53BP1 in repair of IR-induced DNA damage in vertebrate cells, we analyzed the X-ray sensitivity of 53BP1- and various NHEJ-deficient cells. We show that there is a 53BP1-dependent repair pathway that is distinct from the Ku-dependent, and Artemis-dependent pathways. This 53BP1-dependent repair pathway substantially contributes to survival of cells irradiated with IR during G1 phase.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Exon 9 of the two 53BP1 alleles in DT40 cells was targeted for disruption (Fig. 1A). Southern blot analysis using a probe for introns 5 or 10 confirmed the disruption of the 53BP1 alleles (Fig. 1B). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of exons 6 to 9 but not exons 9 to 14 or 9 to 28 in 53BP1 transcripts (Fig. 1C). Because this transcript carried an in-frame stop codon in exon 9, the predicted translation product lacks the C-terminal region, which includes the kinetochore binding (Jullien et al. 2002), DNA-binding, nuclear localization (Iwabuchi et al. 2003) and two BRCT domains (Koonin et al. 1996). Mice expressing the truncated 53BP1, which lacks these C-terminal functional domains, have been reported to show a 53BP1 knockout phenotype (Morales et al. 2003). These data suggest that the 53BP1–/– cells are 53BP1-null DT40 mutants. These 53BP1–/– cells grew with similar kinetics as the wild-type DT40 cells (Fig. 1D).
|
To determine whether 53BP1–/– cells have defects in their responses to IR-induced DNA damage, we examined their X-ray sensitivity using a colony formation assay. Unsynchronous 53BP1–/– and wild-type cells showed similar sensitivity to X-ray (Fig. 2A, left panel). In contrast, as reported previously (Adachi et al. 2001; Tauchi et al. 2002), Lig IV–/– and HR-deficient Nbs1–/–/– cell lines were hypersensitive to X-rays (Fig. 2A, right panel). Also, as previously described (Takata et al. 1998), Ku70–/– cells showed a characteristic biphasic pattern of X-ray sensitivity (Fig. 2A, left panel), reflecting the presence of a resistant subpopulation. Also, as previously described (Takao et al. 1999; Ishiai et al. 2004), ATM–/– and Artemis–/– cells showed modest increases in sensitivity to X-irradiation (Fig. 2A, right panel).
|
Flow cytometric analysis of the DNA content and bromodeoxyuridine (BrdU) uptake following release from mimosine revealed that wild-type, 53BP1–/–, and Lig IV–/– cells progress similarly in the S phase (Fig. 2C). In spite of this, in contrast to Artemis–/–, Ku70–/–, and Lig IV–/– cells, the 53BP1–/– cells showed better resistance to X-rays in early and middle S phases than in the G1 phase. Specifically, as shown in Fig. 2B, the X-ray resistance of 53BP1–/– cells 1 h after release from mimosine treatment (early S phase) recovered to 26% to 30% of the most resistant level (observed 4 h after release, which is during late S phase), and there was a 50% to 65% recovery 2 h after release from mimosine treatment (middle S phase). In contrast, the X-ray resistance of Artemis–/–, Ku70–/–, and Lig IV–/– cells remained at a low level in early and middle S phases and recovered in the late S phase (Fig. 2B). A similar difference in the recovery of X-ray resistance in the early S phase was observed between 53BP1–/– and Lig IV–/– cells synchronized with nocodazole alone (data not shown). These data suggest that 53BP1-deficient cells lack a DNA-damage response that substantially contributes to survival of cells irradiated with IR during the G1 phase.
Intact intra-S and G2/M checkpoints in 53BP1-deficient cells
We next asked whether checkpoint defects contribute to this relatively G1-phase-specific hypersensitivity in 53BP1–/– cells. The intra-S phase checkpoint was examined by measuring radiation-induced inhibition of DNA synthesis 1 h after X-irradiation (Fig. 3A). 53BP1–/– cells were indistinguishable from wild-type cells in the suppression of DNA synthesis by X-irradiation, indicating that the intra-S phase checkpoint is intact in 53BP1–/– cells, whereas intra-S phase checkpoint-deficient Nbs1–/–/– cells showed reduced inhibition of radiation-induced DNA synthesis. With respect to the G2/M checkpoint, we examined the mitotic index at various times after X-irradiation of the cells (Fig. 3B). The fractions of mitotic cells in non-irradiated wild-type and 53BP1–/– cells increased constantly with time after the addition of colcemid. After X-irradiation, most of the wild-type and 53BP1–/– cells did not enter mitosis for several hours, indicating that the G2/M checkpoint is not impaired in 53BP1-deficient cells. The presence of intact intra-S- and G2/M-phase checkpoints in 53BP1-deficient cells is consistent with the idea that the elevated radiosensitivity in the G1 phase is due to a defect in repair rather than the cell cycle checkpoints.
|
It is now widely accepted that most of the lethal effects of IR can be attributed to strand breaks, in particular to DSBs (Friedberg et al. 1995). To investigate the involvement of 53BP1 in HR, one of the major pathways in DSB repair, we examined targeted integration efficiency using the chicken Nbs1 and Rad18 loci (Table 1). A targeting construct was transfected into wild-type and 53BP1–/– cells, and genomic DNA was analyzed by PCR with a set of primers for the antibiotic-resistant marker gene and a site upstream of the 5'-end of the targeting gene in the targeting construct. The targeting frequencies of 53BP1–/– cells were comparable to those of wild-type cells in these two loci, indicating that gene targeting by HR is not impaired in 53BP1–/– cells. Intact HR as a result of gene conversion has been reported in 53BP1-deficient mouse embryonic fibroblasts (Ward et al. 2004).
|
To obtain evidence for a defect in the repair activity in 53BP1–/– cells, we examined chromosomal aberrations (Table 2). Spontaneous chromosomal aberrations, which are frequently observed in DT40 cells deficient in the HR pathway (Sonoda et al. 2001), were not observed in either wild-type or 53BP1–/– cells, providing further support for the idea that 53BP1 is not involved in HR; however, there were more radiation-induced chromosomal aberrations in 53BP1–/– cells than in wild-type DT40 cells. The most significant difference was an elevated frequency of isochromatid-type breaks and gaps in 53BP1–/– cells irradiated during G1-early S phase (6–9 h) (Table 2). The increase in isochromatid-type aberrations suggest that 53BP1 is involved in a repair pathway that plays a major role during G1-early S phase (Takata et al. 1998).
|
-H2AX foci in 53BP1-deficient cells
Phosphorylation of H2AX, a variant form of the histone H2A, is an early response to DSBs (Rogakou et al. 1998). Phosphorylated H2AX (-H2AX) can be observed by immunofluorescence microscopy as discrete nuclear foci. Recent studies indicate that there is a correlation between the number of foci and the number of DSBs induced by IR and that the rate of disappearance of foci reflects the cellular activity of DSB repair, especially in non-replicating, G1-arrested cells (Rothkamm & Lobrich 2003). Although foci are also formed at single strand DNA sites in an ATR (ataxia-telangiectasia-Rad3-related)-dependent manner (Ward & Chen 2001; Limoli et al. 2002), measurement of the rate at which foci disappear is also useful in replicating cells for monitoring their ability to repair DSBs induced by IR (Rothkamm et al. 2003). To obtain further evidence for a defect in the repair activity in 53BP1–/– cells, we performed immunofluorescence analysis of the -H2AX focus induced by X-ray in 53BP1–/– cells (Fig. 4). G1-synchronized cells were released into the cell cycle and subsequently irradiated with 1 Gy of X-rays. We then counted the number of -H2AX foci at various time points. Without irradiation, G1-synchronized wild-type, 53BP1–/–, Ku70–/–, and Artemis–/– cells showed 7.6, 6.8, 8.5, and 8.1 -H2AX foci per cell, respectively. In all cell-lines, the number of -H2AX foci 30 min after 1 Gy of X-irradiation increased to as many as 20 per cell (Fig. 4). Because flow cytometry showed that DT40 cells contain approximately half the amount of DNA as human fibroblasts (data not shown), 15 X-ray-induced -H2AX foci in DT40 cells is comparable to the 35 initial DSBs per Gy reported in human fibroblasts (Lobrich et al. 1993). Ku70–/– cells were markedly delayed in disappearance of -H2AX foci, confirming that the core NHEJ repairs the majority of IR-induced DSBs. There were fewer foci persisting 8 h after irradiation in 53BP1–/– and Artemis–/– cells than in Ku70–/– cells but substantially more than in wild-type cells. These findings suggest that 53BP1–/– cells have defects in the repair of IR-induced DSBs. The number of foci persisting 8 h after irradiation roughly correlated with the radiosensitivities of cells irradiated in the G1 phase (Figs 4 and 5A,B).
|
To determine whether 53BP1 functions in the core NHEJ pathway, we carried out epistasis-type analysis using 53BP1–/– and Ku70–/– cells as well as cells deficient in both 53BP1 and Ku70 (53BP1–/– Ku70–/– cells) (Fig. 5A). When cells were irradiated in G1 phase, 53BP1–/– and Ku70–/– cells were more sensitive to irradiation than wild-type cells, and Ku70–/– cells were more sensitive than 53BP1–/– cells. In addition, 53BP1–/– Ku70–/– cells were more sensitive than either of the single mutants. Thus, 53BP1 and Ku70 are nonepistatic in cell survival, suggesting the existence of a 53BP1-dependent pathway that is independent of the core NHEJ pathway.
|
We next asked whether the 53BP1-dependent pathway requires Lig IV (Fig. 5D). We found that 53BP1–/– Lig IV–/– cells had the same degree of X-ray sensitivity as Lig IV–/– cells. The epistasis-type analysis therefore strongly suggests that Lig IV is required for the 53BP1 dependent-repair pathway.
53BP1-dependent repair pathway is resistant to wortmannin
Wortmannin is an inhibitor of the phosphatidylinositol 3-kinase family, which includes DNA-PK (Sarkaria et al. 1998). It is thought that DSB ends are recognized by the Ku70/Ku80 heterodimer, which then recruits DNA-PKcs to sites of DNA DSBs, after which the kinase activity of DNA-PK is stimulated by association with DNA ends (Lieber et al. 2003; Downs & Jackson 2004). To confirm importance of the 53BP1-dependent repair pathway in cell survival, we next examined the effect of wortmannin on the X-ray sensitivity of cells receiving 1 Gy of X-rays at the G1 phase (Fig. 6A, left panel).
|
Finally, to determine whether damaged DNA remained unrepaired in wortmannin-treated 53BP1–/– cells, we examined the effect of wortmannin on disappearance of -H2AX foci after X-irradiation (Fig. 6B). G1-synchronized cells were released into the cell cycle and subsequently irradiated with 1 Gy of X-rays in the presence or absence of wortmannin. More than 20 -H2AX foci per cell were observed 30 min after irradiation in both wild-type and 53BP1–/– cells. At 4 h after irradiation, the number of foci per cell decreased to 15.0, 16.7 and 18.2 in wortmannin-untreated, wortmannin-treated wild-type and wortmannin-untreated 53BP1–/– cells, respectively. However, wortmannin-treated 53BP1–/– cells showed 24.4 foci per cell at 4 h after irradiation. These data are consistent with the hypersensitivity to X-ray of wortmannin-treated 53BP1–/– cells, and suggest that damaged DNA remains unrepaired in wortmannin-treated 53BP1–/– cells.
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
s BRCT repeats (Iwabuchi et al. 1994, 1998). 53BP1 is thought to be a human counterpart of Saccharomyces cerevisiae checkpoint protein Rad9 because of the similarity in their BRCT repeats (Koonin et al. 1996). This possibility is supported by accumulating evidence that 53BP1 plays an important role in checkpoint signaling (Mochan et al. 2004). In a previous study, we observed that the 53BP1 domain responsible for focus formation enhances the ligation reaction catalyzed by recombinant Lig IV/Xrcc4 (Iwabuchi et al. 2003). In this study, we show that 53BP1 plays a role in repair of IR-induced DNA damage in a pathway distinct from the core NHEJ and the ATM/Artemis-dependent pathways, and that the 53BP1-dependent repair pathway contributes to survival of cells irradiated with IR during the G1 phase of the cell cycle. 53BP1 may be involved in repair of DNA damage induced by IR
53BP1–/– cells exhibited increased radiosensitivity in the G1 phase. This may be due to defective repair for X-ray-induced DNA damage, because the 53BP1–/– cells have apparently normal intra-S and G2/M checkpoints. Because HR plays an essential role in repair of DSBs induced by replication processes, DT40 cells deficient in the HR pathway frequently show an increase in spontaneous chromosomal breaks (Sonoda et al. 2001). Such spontaneous chromosomal aberrations, however, were not observed in 53BP1-deficient cells. Furthermore, gene targeting by HR is not impaired in 53BP1-deficient cells. These results suggest that 53BP1 is not required for HR. Rather, several of our findings suggest the involvement of 53BP1 in NHEJ:
However, the possibility is not excluded that 53BP1 is involved in the suppression of IR-induced apoptosis or repair of other IR-induced DNA damage than DSBs, for example single-strand breaks, DNA-protein cross-links and various base damages.
53BP1 plays a role in a pathway that is distinct from both the core NHEJ and ATM/Artemis-dependent pathways
We propose that 53BP1 functions in repair of IR-induced DNA damage in a pathway distinct from both the core NHEJ and ATM/Artemis-dependent pathways in the G1 phase cells (Fig. 7). The core NHEJ (Riballo et al. 2004) (pathway A in Fig. 7) is well-known and involves the Ku70/Ku80 heterodimer, DNA-PKcs, and the Lig IV/Xrcc4 complex. In mammalian cells, the majority of DSBs induced by IR in G1-phase cells are rejoined rapidly by the core NHEJ, whereas approximately 10% of the DSBs are repaired over a prolonged time. Recently, it was reported that this slower end-joining process requires ATM and Artemis (Riballo et al. 2004). Artemis–/– DT40 cells show increased radiosensitivity in the G1 and early S phase, supporting the involvement of Artemis in NHEJ. Furthermore, our epistasis-type analysis supports the idea that Artemis plays a role in a common pathway with ATM.
|
The increase in the radiosensitivity of wild-type, ATM–/–, and 53BP1–/– cells by wortmannin could be due to inhibition of both the core NHEJ and Artemis-dependent pathways. However, the ability of wortmannin-treated wild-type and ATM–/– cells but not 53BP1–/– cells to form colonies indicates that there is a pathway that is dependent on 53BP1 and resistant to wortmannin (pathway C in Fig. 7). In 53BP1–/– mammalian cells, addition of inhibitors of ATM or DNA-PK does not affect DSB repair activity (Riballo et al. 2004). This contradicts our results in DT40 cells. The discrepancy may be due to redundancy between DNA-PK and ATM in the modulation of Artemis, as discussed above. Importantly, since colonies are formed by wortmannin-treated wild-type cells, the 53BP1-dependent pathway may be the third pathway in NHEJ. Alternatively, 53BP1 may play dual roles in repair of IR-induced DNA damage: roles in repair of DSBs, and in repair of other DNA damages than DSBs. In this model, 53BP1 may function in the Artemis-dependent NHEJ pathway, as suggested by Riballo et al. in mammalian cells (Riballo et al. 2004).
In Artemis–/– cells, the core NHEJ and 53BP1-dependent pathways are functional, and wortmannin inhibits the core NHEJ pathway, resulting in a reduction in the number of colonies. However, in the presence of wortmannin, Artemis–/– cells are more sensitive than wild-type cells to X-rays, suggesting that Artemis has a DNA-PK-independent function. Wortmannin-treated Ku70–/– cells are more sensitive to X-rays than wortmannin-treated wild-type cells. This suggests that the Ku70/Ku80 complex also has a DNA-PK-independent function.
53BP1 may function as a backup pathway towards the Lig IV/Xrcc4 complex
Colony formation in Lig IV–/– cells suggests the presence of a Lig IV-independent end-joining pathway. The severe radiosensitivity of wortmannin-treated 53BP1–/– cells suggests that Lig IV-independent repair does not function in these cells. In Lig IV–/– cells, the absence of Lig IV may allow access of other DNA ligase(s) to sites of ligation reaction. The DNA-PKcs protein but not the kinase activity of DNA-PK is required for recruitment of the Lig IV/Xrcc4 complex to chromatin (Drouet et al. 2005). In contrast, the DNA-PK kinase activity is required for functional end-joining in DSB repair (Chan et al. 2002). In wortmannin-treated cells, even though the Lig IV/Xrcc4 complex is present at ligation sites, the ligation reaction might be inhibited due to the lack of upstream signals produced by DNA-PK kinase activity. Thus, the severe radiosensitivity of wortmannin-treated 53BP1–/– cells might be due to the defect in the 53BP1-dependent pathway as well as the inability of other DNA ligases to access sites of the ligation reaction due to the binding of non-functional Lig IV/Xrcc4 complexes. 53BP1 may function as a backup pathway towards the Lig IV/Xrcc4 complex, when upstream phosphorylation signals for this complex are weak or absent.
Finally, the 53BP1-dependent pathway made a larger contribution to cell survival in G1 than in early S phase, suggesting that the 53BP1-dependent pathway is regulated at the G1 to S phase transition by mechanisms distinct from the other two pathways. Recently, it has been shown that 53BP1-deficient mice have intact V(D)J recombination but impaired class switch recombination (Manis et al. 2004; Ward et al. 2004). It is unclear whether the 53BP1-dependent repair pathway identified in this study is involved in class switch recombination. If, as proposed, class switch recombination occurs in the G1 phase of the cell cycle (Petersen et al. 2001), it is possible that, in vertebrates, class switch recombination is the main stage at which 53BP1 participates in DNA damage repair.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Sequence information for chicken 53BP1 genomic DNA and cDNA was obtained from GENBANK (accession no. AADN01085436) and the Biotechnology and Biological Sciences Research Council (BBSRC) ChickEST Database (sequence ID 055102.1 for exons 6 to 10, 350592.1 for exons 12 to 14, and 356837.3 for exons 21 to 28), respectively. Sequence information for chicken Ku70 genomic DNA was obtained from GENBANK (accession no. NW-060209). The genomic DNA fragment for exons 6–10 of the chicken 53BP1 was generated by PCR using genomic DNA extracted from wild-type DT40 cells. Targeting constructs for 53BP1 were generated by inserting a drug selection marker gene at the BamHI site in exon 9 of the 53BP1 genomic DNA fragment. Targeting constructs for Ku70 were designed as previously described (Takata et al. 1998).
Cell culture and colony formation assay
Cells were cultured in RPMI1640 medium (Nissui) supplemented with 10% fetal calf serum (Valley Biomedical Inc.), 1% chicken serum (Medical and Biological Laboratories), and 10 µM ß-mercaptoethanol at 39.5 °C. DNA transfection and selection were performed as previously described (Buerstedde & Takeda 1991). X-irradiation at 150 kV and 20 mA was performed at a dose rate of 1.9 Gy/min with an X-ray irradiator (Hitachi Medico). The colony formation assay was performed as previously described (Hashimoto et al. 2003). Briefly, an appropriate number of cells were plated in 1.5% methylcellulose (Sigma)-containing medium immediately after irradiation. After incubation at 39.5 °C for 7 days, the surviving cell fractions were calculated by comparing the numbers of colonies formed in the irradiated cultures with those in a non-irradiated control. For wortmannin treatment, cultures were adjusted to 10 µM wortmannin (Sigma) just before receiving 1 Gy of irradiation. After irradiation, cells were cultured for 1 h at 39.5 °C, washed three times with phosphate-buffered saline (PBS) containing 5% calf serum (GibcoBRL), and subsequently seeded on the methylcellulose plates. For a non-irradiated control, cells treated with wortmannin were cultured for 1 h at 39.5 °C without irradiation, washed three times as described above and subsequently seeded on the methylcellulose plates. After incubation at 39.5 °C for 7 days, the surviving cell fractions were calculated as described above.
Chromosomal aberration assay
The chromosomal aberration assay was performed as previously described (Takata et al. 1998). Briefly, 1.0 µg/mL of colcemid (Sigma) was added in cultures 3 h before cell harvest. Harvested cells were incubated in 1 mL of 0.9% sodium citrate for 15 min at room temperature and fixed in 5 mL of freshly prepared 3 : 1 mixture of methanol and acetic acid. The cell suspension was dropped on to an ice-cold wet glass slide and immediately flame dried. Slides were stained with 3% Giemsa solution at pH 6.4 for 10 min.
Cell cycle synchronization
To enrich cells in prometaphase, cells were cultured in medium containing 1.0 µg/mL nocodazole (Sigma) for 8 h. For G1 phase synchronization, cells treated with nocodazole were washed three times with PBS containing 5% calf serum and then cultured in medium containing 0.8 mM mimosine (Sigma) for 8 h. Synchronized cells were washed three times as described above and cultured in medium to release them into the cell cycle.
Flow cytometric analysis and DNA synthesis assay
Cells were labeled for 15 min with 10 µM BrdU (Sigma). Cells were then harvested and fixed with 70% ethanol at 4 °C. Fixed cells were incubated as follows: (i) 4 N HCl containing 0.5% Triton X-100 for 30 min at room temperature (ii) fluorescein isothiocyanate-conjugated anti-BrdU antibody (Becton Dickinson) for 1 h at room temperature, and (iii) 5 µg/mL propidium iodide in PBS for 10 min at room temperature. After each incubation, cells were washed with PBS containing 1% bovine serum albumin. Flow cytometric analysis was performed using a FACScan (Becton Dickinson), and data were displayed using Cell Quest software (Becton Dickinson). DNA synthesis assays were performed as previously described (Tauchi et al. 2002) with the following minor modifications: cells were irradiated with X-rays, incubated for 1 h, and labeled with [3H]thymidine for 30 min.
G2/M checkpoint assay and -H2AX staining
In the G2/M checkpoint assay, non-irradiated or irradiated cells were cultured in medium containing 0.1 µg/mL colcemid and collected at 1-h intervals. Cells were attached to the surface of a slide glass by centrifugation, fixed in 4% paraformaldehyde for 10 min, permeabilized in PBS containing 1% Nonidet P-40, and stained with 4',6'-diamino-2-phenylindole. To determine the percentage of mitotic cells (mitotic index), more than 200 nuclei were counted using a fluorescence microscope (Olympus). For -H2AX staining, cells were fixed as described above and then treated with 70% ethanol for 10 min. After a brief rinse with PBS, cells were permeabilized as described above, blocked in 3% bovine serum albumin at room temperature for 20 min, and then incubated with anti--H2AX antibody (2 µg/mL, Upstate, clone JBW301) at room temperature for 1 h. After extensive washing, cells were incubated with tetramethylrhodamine isothiocyanate-conjugated secondary antibody (Dako) at room temperature for 1 h. Cells were counterstained with 4',6'-diamino-2-phenylindole, and foci of nonapoptotic nuclei were counted by eye using a fluorescence microscope with an oil-immersion objective lens (PlanApo 100X).
|
Acknowledgements |
|---|
|
Footnotes |
|---|
* Correspondence: E-mail: kuni-kmu{at}kanazawa-med.ac.jp
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Anderson, L., Henderson, C. & Adachi, Y. (2001) Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol. Cell. Biol.
21, 1719–1729.
Buerstedde, J.M. & Takeda, S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67, 179–188.[CrossRef][Medline]
Chan, D.W., Chen, B.P., Prithivirajsingh, S., et al. (2002) Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev.
16, 2333–2338.
DiTullio, R.A., Mochan, T.A., Venere, M., et al. (2002) 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer. Nat. Cell Biol. 12, 998–1002.
Downs, J.A. & Jackson, S.P. (2004) A means to a DNA end: the many roles of Ku. Nat. Rev. Mol. Cell Biol. 5, 367–378.[CrossRef][Medline]
Drouet, J., Delteil, C., Lefrancois, J., Concannon, P., Salles, B. & Calsou, P. (2005) DNA-dependent protein kinase and XRCC4-DNA ligase IV mobilization in the cell in response to DNA double strand breaks. J. Biol. Chem.
280, 7060–7069.
Fernandez-Capetillo, O., Chen, H.T., Celeste, A., et al. (2002) DNA damage-induced G2/M checkpoint activation by histone H2AX and 53BP1. Nat. Cell Biol. 12, 993–997.
Friedberg, E.C., Walker, G.C. & Siede, W. (1995) DNA damage. In: DNA Repair and Mutagenesis (eds E.C. Friedberg, G.C. Walker & W. Siede), pp. 1–58. Washington, D.C.: American Society for Microbiology.
Hashimoto, M., Rao, S., Tokuno, O., et al. (2003) DNA-PK: the major target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via NHEJ pathway. J. Radiat. Res. 44, 151–159.[Medline]
Ishiai, M., Kimura, M., Namikoshi, K., et al. (2004) DNA cross-link repair protein SNM1A interacts with PIAS1 in nuclear focus formation. Mol. Cell. Biol.
24, 10733–10741.
Iwabuchi, K., Bartel, P.L., Li, B., Marraccino, R. & Fields, S. (1994) Two cellular proteins that bind to wild-type but not mutant p53. Proc. Natl. Acad. Sci. USA
91, 6098–6102.
Iwabuchi, K., Basu, B.P., Kysela, B., et al. (2003) Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA. J. Biol. Chem.
278, 36487–36495.
Iwabuchi, K., Li, B., Massa, H.F., Trask, B.J., Date, T. & Fields, S. (1998) Stimulation of p53-mediated transcriptional activation by the p53-binding proteins, 53BP1 and 53BP2. J. Biol. Chem.
273, 26061–26068.
Jackson, S.P. (2002) Sensing and repairing DNA double-strand breaks. Carcinogenesis
23, 687–696.
Jeggo, P.A. (1998) DNA breakage and repair. Adv. Genet. 38, 185–211.[Medline]
Johnson, R.D. & Jasin, M. (2000) Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells. EMBO J. 19, 3398–3407.[CrossRef][Medline]
Jullien, D., Vagnarelli, P., Earnshaw, W.C. & Adachi, Y. (2002) Kinetochore localisation of the DNA damage response component 53BP1 during mitosis. J. Cell Sci.
115, 71–79.
Koonin, E.V., Altschul, S.F. & Bork, P. (1996) BRCA1 protein products ... Functional motifs.... Nat. Genet. 13, 266–268.[CrossRef][Medline]
Lieber, M.R., Ma, Y., Pannicke, U. & Schwarz, K. (2003) Mechanism and regulation of human non-homologous DNA end-joining. Nat. Rev. Mol. Cell Biol. 4, 712–720.[CrossRef][Medline]
Limoli, C.L., Giedzinski, E., Bonner, W.M. & Cleaver, J.E. (2002) UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, -H2AX formation, and Mre11 relocalization. Proc. Natl. Acad. Sci. USA
99, 233–238.
Lobrich, M., Ikpeme, S., Haub, P., Weber, K.J. & Kiefer, J. (1993) DNA double-strand break induction in yeast by X-rays and alpha-particles measured by pulsed-field gel electrophoresis. Int. J. Radiat. Biol. 64, 539–546.[Medline]
Lobrich, M. & Jeggo, P.A. (2005) Harmonising the response to DSBs: a new string in the ATM bow. DNA Repair (Amst.) 4, 749–759.[CrossRef][Medline]
Ma, Y., Pannicke, U., Schwarz, K. & Lieber, M.R. (2002) Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 781–794.[CrossRef][Medline]
Manis, J.P., Morales, J.C., Xia, Z., Kutok, J.L., Alt, F.W. & Carpenter, P.B. (2004) 53BP1 links DNA damage-response pathway to immunoglobulin heavy chain class-switch recombination. Nat. Immunol. 5, 481–487.[CrossRef][Medline]
Mochan, T.A., Venere, M., DiTullio, R.A. & Halazonetis, T.D. (2004) 53BP1, an activator of ATM in response to DNA damage. DNA Repair (Amst.) 3, 945–952.[CrossRef][Medline]
Morales, J.C., Xia, Z., Lu, T., et al. (2003) Role for BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability. J. Biol. Chem.
278, 14971–14977.
Moshous, D., Callebaut, I., de Chasseval, R., et al. (2001) Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 105, 177–186.[CrossRef][Medline]
Petersen, S., Casellas, R., Reina-San-Martin, B., et al. (2001) AID is required to initiate Nbs1/-H2AX focus formation and mutations at sites of class switching. Nature
414, 660–665.[CrossRef][Medline]
Rappold, I., Iwabuchi, K., Date, T. & Chen, J. (2001) Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways. J. Cell Biol.
153, 613–620.
Riballo, E., Kuhne, M., Rief, N., et al. (2004) A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to -H2AX foci. Mol. Cell
16, 715–724.[CrossRef][Medline]
Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem.
273, 5858–5868.
Rothkamm, K., Kruger, I., Thompson, L.H. & Lobrich, M. (2003) Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol. Cell. Biol.
23, 5706–5715.
Rothkamm, K. & Lobrich, M. (2003) Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc. Natl. Acad. Sci. USA
100, 5057–5062.
Sarkaria, J.N., Tibbetts, R.S., Busby, E.C., Kennedy, A.P., Hill, D.E. & Abraham, R.T. (1998) Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res.
58, 4375–4382.
Schultz, L.B., Chehab, N.H., Malikzay, A. & Halazonetis, T.D. (2000) p53-binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J. Cell Biol.
151, 1381–1390.
Shiloh, Y. (2003) ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168.[CrossRef][Medline]
Sonoda, E., Takata, M., Yamashita, Y.M., Morrison, C. & Takeda, S. (2001) Homologous DNA recombination in vertebrate cells. Proc. Natl. Acad. Sci. USA
98, 8388–8394.
Takao, N., Kato, H., Mori, R., et al. (1999) Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation. Oncogene 18, 7002–7009.[CrossRef][Medline]
Takata, M., Sasaki, S., Sonoda, E., et al. (1998) Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17, 5497–5508.[CrossRef][Medline]
Tauchi, H., Kobayashi, J., Morishima, K., et al. (2002) Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420, 93–98.[CrossRef][Medline]
Wang, B., Matsuoka, S., Carpenter, P.B. & Elledge, S.J. (2002) 53BP1, a mediator of the DNA damage checkpoint. Science
298, 1435–1438.
Ward, I.M. & Chen, J. (2001) Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem.
276, 47759–47762.
Ward, I.M., Reina-San-Martin, B., Olaru, A., et al. (2004) 53BP1 is required for class switch recombination. J. Cell Biol.
165, 459–464.
Xia, Z., Morales, J.C., Dunphy, W.G. & Carpenter, P.B. (2000) Negative cell cycle regulation and DNA-damage inducible phosphorylation of the BRCT protein 53BP1. J. Biol. Chem. 276, 2708–2718.[Medline]
Received: 28 February 2006
Accepted: 11 May 2006
This article has been cited by other articles:
|
|
 |
|
  Y. Kang, J.-H. Lee, N. N. Hoan, H.-M. Sohn, I.-Y. Chang, and H. J. You Protein Phosphatase 5 Regulates the Function of 53BP1 after Neocarzinostatin-induced DNA Damage J. Biol. Chem., April 10, 2009; 284(15): 9845 - 9853. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Watanabe, K. Iwabuchi, J. Sun, Y. Tsuji, T. Tani, K. Tokunaga, T. Date, M. Hashimoto, M. Yamaizumi, and S. Tateishi RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1 Nucleic Acids Res., April 1, 2009; 37(7): 2176 - 2193. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
)or absence (
) of wortmannin, and subsequently subjected to a colony formation assay. Colony formation was not observed in wortmannin-treated 53BP1–/– cell culture (*), indicating that the surviving cell fraction was less than 0.01%. Results represent the means ± SD of three independent experiments. (B) Effect of wortmannin on disappearance of 
) after irradiation. The mean number of foci per cell ± standard error is shown from the analysis of more than 40 nuclei.