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Recruitment of ATM Protein to Double Strand DNA Irradiated with Ionizing Radiation

1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês

10.1074/jbc.274.36.25571

1083-351X

Keiji Suzuki, Seiji Kodama, Masami Watanabe,

Radiation Therapy and Dosimetry

The product of the ATM gene, which is mutated in ataxia telangiectasia, is a nuclear phosphoprotein, and it involves the activation of the p53 pathway after ionizing radiation. Here we show that the ATM protein is constitutively associated with double strand DNA and that the interaction increases when the DNA is exposed to ionizing radiation. The ATM protein also had affinity to restriction endonuclease Pvu II-digested DNA, but not to UV-irradiated DNA nor X-irradiated single-stranded DNA. The immunoprecipitation experiment detected very weak association between ATM and DNA-PK proteins, and immunodepletion of DNA-PK showed little or no effect on the interaction of the ATM protein with damaged DNA, indicating that an interaction with DNA-PK might not be required for the recruitment of the ATM protein to damaged DNA. Furthermore, the association was also confirmed in xrs-5 and xrs-6e cells, which are Chinese hamster ovary mutant cell lines defective in Ku80 function. These results indicate that the ATM protein is recruited to the site of DNA damage and it recognizes double strand breaks by itself or through an association with other DNA-binding protein other than DNA-PK and Ku80 proteins. The product of the ATM gene, which is mutated in ataxia telangiectasia, is a nuclear phosphoprotein, and it involves the activation of the p53 pathway after ionizing radiation. Here we show that the ATM protein is constitutively associated with double strand DNA and that the interaction increases when the DNA is exposed to ionizing radiation. The ATM protein also had affinity to restriction endonuclease Pvu II-digested DNA, but not to UV-irradiated DNA nor X-irradiated single-stranded DNA. The immunoprecipitation experiment detected very weak association between ATM and DNA-PK proteins, and immunodepletion of DNA-PK showed little or no effect on the interaction of the ATM protein with damaged DNA, indicating that an interaction with DNA-PK might not be required for the recruitment of the ATM protein to damaged DNA. Furthermore, the association was also confirmed in xrs-5 and xrs-6e cells, which are Chinese hamster ovary mutant cell lines defective in Ku80 function. These results indicate that the ATM protein is recruited to the site of DNA damage and it recognizes double strand breaks by itself or through an association with other DNA-binding protein other than DNA-PK and Ku80 proteins. ataxia telangiectasia gray radioimmune precipitation buffer Chinese hamster ovary Ataxia telangiectasia (AT)1 is a human autosomal recessive disorder characterized by a variety of clinical symptoms. At the cellular level, AT cells exhibit hypersensitivity to ionizing radiation, radio-resistant DNA synthesis, and a high frequency of chromosome aberrations (reviewed in Refs. 1Jachson S.P. Curr. Biol. 1995; 5: 1210-1212Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 2Meyn M.S. Cancer Res. 1995; 55: 5991-6001PubMed Google Scholar, 3Shiloh Y. Eur. J. Hum. Genet. 1995; 3: 116-138Crossref PubMed Scopus (154) Google Scholar). Recently, the gene defective in AT cells has been cloned and designated ATM (4Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.C. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2396) Google Scholar). Sequence analysis revealed that the ATM protein shows homology to a family related to phosphatidylinositol 3-kinase, those include phosphatidylinositol 3-kinase p110 subunit, FRAP, RAFT1, and DNA-PK proteins in mammalian cells (2Meyn M.S. Cancer Res. 1995; 55: 5991-6001PubMed Google Scholar, 5Hartley K.O. Gell D. Smith G.C.M. Zhang H. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Anderson C.W. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 6Savitsky K. Sfez S. Tagle D.A. Ziv Y. Sartiel A. Collins F.C. Shiloh Y. Rotman G. Hum. Mol. Genet. 1995; 4: 2025-2032Crossref PubMed Scopus (481) Google Scholar, 7Zakian V.A. Cell. 1995; 82: 685-687Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Because p53 accumulation is diminished or severely delayed in AT cells irradiated with ionizing radiation, ATM protein has been suggested to be involved in p53 activation (8Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2958) Google Scholar, 9Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (784) Google Scholar, 10Khanna K.K. Lavin M.F. Oncogene. 1993; 8: 3307-3312PubMed Google Scholar, 11Canman C.E. Wolff A.C. Chen C.-Y. Fornace Jr., A.J. Kastan M.B. Cancer Res. 1994; 54: 5054-5058PubMed Google Scholar). p53 is a nuclear protein whose phosphorylation is required for its stability and activity (reviewed in Refs. 12Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6804) Google Scholar, 13Agarwal M.L. Taylor W.R. Chernov M.V. Chernova O.B. Stark G.R. J. Biol. Chem. 1998; 273: 1-4Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, 14Meek D.W. Cell. Signal. 1998; 10: 159-166Crossref PubMed Scopus (179) Google Scholar, 15Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). Our study and others (16Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 17Ghosh J.C. Suzuki K. Kodama S. Watanabe M. J. Radiat. Res. 1999; 41: 23-37Crossref Scopus (20) Google Scholar) have reported that ionizing radiation causes p53 protein accumulation, and recently phosphorylation of p53 at serine 15 has been shown to control p53 accumulation through inhibition of its interaction with MDM2 (18Shieh S.-Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar, 19Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3790) Google Scholar, 20Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2880) Google Scholar). Furthermore, recent in vitro experiments have indicated that ATM phosphorylates p53 protein at serine 15 (21Banin S. Moyal L. Shieh S.-Y. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1722) Google Scholar, 22Canman C.E. Lim D.-S. Cimprivh K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1722) Google Scholar). Although there has been a report indicating the phosphorylation of serine 15 in AT cells (23Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (715) Google Scholar), it is highly possible that the activity of p53 protein is mediated by ATM kinase activity. Very little is known about the mechanism by which ATM protein is activated by DNA damage. Previous studies have shown that ATM protein levels do not change during cell cycle progression or after exposure to ionizing radiation (24Lakin N.D. Weber P. Stankovic T. Rottinghaus S.T. Taylor A.M.R. Jackson S.P. Oncogene. 1996; 13: 2707-2716PubMed Google Scholar, 25Brown K.D. Ziv Y. Sadanandan S.N. Chessa L. Collins F.S. Shiloh Y. Tangle D.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1840-1845Crossref PubMed Scopus (150) Google Scholar, 26Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Zhang N. Farrell A. Ramsey J. Gatti R. Lavin M. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (170) Google Scholar), and therefore, it has been hypothesized that ATM protein may not be an inducible effector, but may act as a transducer or, more likely, a sensor for DNA damage. Previous studies have indicated that ATM protein harbors a putative leucine zipper motif (6Savitsky K. Sfez S. Tagle D.A. Ziv Y. Sartiel A. Collins F.C. Shiloh Y. Rotman G. Hum. Mol. Genet. 1995; 4: 2025-2032Crossref PubMed Scopus (481) Google Scholar), whose structure is commonly detected in DNA binding proteins that form homo- or heterodimers. Furthermore, the association of ATM protein with meiotic chromosomes has been reported, indicating that the ATM protein may sense double strand break related structures occurring in synapsing meiotic chromosomes (27Keegan K.S. Holtzman D.A. Plug A.W. Christenson E.R. Brainerd E.E. Flaggs G. Bentley N.J. Taylor E.M. Meyn M.S. Moss S.B. Carr A.M. Ashley T. Hoekstra M.F. Gene Dev. 1996; 10: 2423-2437Crossref PubMed Scopus (254) Google Scholar). Thus, it is possible that ATM protein also interacts with DNA double strand breaks induced by ionizing radiation. Because DNA-PK, a protein kinase that shares amino acid homology with ATM, has been shown to be recruited through its association with Ku70/80 proteins bound to the DNA ends of double strand breaks, and DNA-PK has a putative leucine zipper motif (5Hartley K.O. Gell D. Smith G.C.M. Zhang H. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Anderson C.W. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 28Suwa A. Hirakata M. Takeda Y. Jesch S.A. Mimori T. Hardin J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6904-6908Crossref PubMed Scopus (171) Google Scholar), it can be hypothesized that ATM protein interacts with damaged DNA by itself or through an association with DNA-PK or Ku-like proteins. In the present study, we examined whether ATM protein interacts with damaged DNA in vitro. Our results show that the ATM protein constitutively associates with double strand DNA and that its interaction is potentiated when the DNA is exposed to ionizing radiation. The ATM protein also interacted with Pvu II-treated DNA but not with DNA exposed to UVC light. Although a very weak interaction between ATM and DNA-PK was observed, immunodepletion of DNA-PK protein did not affect the interaction of ATM protein with damaged DNA. Furthermore, ATM associated with damaged DNA in Chinese hamster cells deficient in Ku80. These results suggested that association of the ATM protein with damaged DNA is independent of DNA-PK or Ku80 proteins. Normal human embryo cells (HE49) were cultured in minimum Eagle's medium supplemented with 10% fetal bovine serum (Trace Bioscience PTY Ltd., Australia) as described previously (30Watanabe M. Suzuki M. Suzuki K. Nakano K. Watanabe K. Int. J. Radiat. Biol. 1992; 62: 711-718Crossref PubMed Scopus (54) Google Scholar). 1 × 106 cells were seeded in T75 flasks (75 cm2) and subcultured every 4–5 days to maintain exponential cell growth. AT5BI and AT2KY cells were obtained from Japanese Cancer Research Resources Bank. The xrs-5 cells and xrs-6e cells were obtained from Dr. Tom K. Hei, Columbia University, New York and from Dr. William F. Morgan, University of California San Fransisco, respectively. Exponentially growing cells were irradiated with x-rays from a x-ray generator at 150 kVp and 5 mA with a 0.1-mm copper filter. The dose rate was 0.44 Gy/min. Ultraviolet light was exposed by UV-light box (Ultraviolet Products, Upland, CA) equipped with two germicidal lamps. DNA-cellulose was irradiated with x-rays in RIPA buffer or exposed to UV under semidried condition. Cells were lysed in RIPA buffer (50 mm Tris-HCl, pH 7.2, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing 1 mm4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. The cell lysate was cleared by centrifugation at 15,000 rpm for 10 min at 4 °C, and the supernatant was used as total cellular protein (31Suzuki K. Kodama S. Watanabe M. Radiat. Res. 1998; 149: 195-201Crossref PubMed Scopus (22) Google Scholar). Protein concentration was determined by BCA protein assay (Pierce). For extraction of the nuclear protein, cells were lysed in Lysis buffer (10 mm HEPES, pH 8.0, 50 mm NaCl, 0.5 msucrose, 0.1 mm EDTA, 0.5% Triton X-100, 1 mmdithiothreitol, 5 mm MgCl2) as described previously (31Suzuki K. Kodama S. Watanabe M. Radiat. Res. 1998; 149: 195-201Crossref PubMed Scopus (22) Google Scholar). The lysate was centrifuged at 5000 rpm for 1 min and the supernatant was collected as cytoplasmic protein. The resultant pellet was washed repeatedly with Lysis buffer, suspended in Lysis buffer containing 0.5 m NaCl and 5 mmspermidine, and incubated for 30 min at 4 °C. The lysate was cleared by centrifugation at 12,000 rpm for 30 min at 4 °C, and the supernatant was used as nuclear protein. Protein concentration was determined with a protein assay kit (Bio-Rad, Tokyo, Japan). Sixteen to thirty-two μg of total or nuclear proteins were electrophoresed on SDS-polyacrylamide gels as described (31Suzuki K. Kodama S. Watanabe M. Radiat. Res. 1998; 149: 195-201Crossref PubMed Scopus (22) Google Scholar). The proteins were electrophoretically transferred to polyvinyl difluoride membrane in transfer buffer (100 mm Tris, 192 mm glycine), and the membrane was incubated with blocking solution (10% skim milk) overnight. The membrane was incubated with anti-ATM mouse monoclonal antibody (clone 1A1, Gene Tex, San Antonio, TX), anti-DNA-PK mouse monoclonal antibody mixture (MC-365, Kamiya Biomedical Co., Seattle, WA), and anti-p53 monoclonal antibody (clone BP53–12, Lab Vision Corp., Fremont, CA) for 2 h. The membrane was then incubated with biotinylated secondary antibody followed by streptavidin-alkalinephophatase. The band was visualized after addition of nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as a substrate. Total cell lysates (500 μg) were incubated with native or denatured DNA-cellulose (Amersham Pharmacia Biotech, Tokyo) equilibrated in RIPA buffer (50 mmTris-HCl, pH 7.2, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS). Native double-stranded DNA-cellulose was irradiated with 20 Gy of x-rays, 5000 J/m2 UV light, or 1000 units of Pvu II for 4 h. The cell lysates were also incubated with native DNA-cellulose (75 μg of DNA) equilibrated in RIPA buffer containing between 150 and 550 mm NaCl. For DNA-PK- or ATM-depleted samples the cell lysate was incubated with monoclonal antibodies against DNA-PK or ATM for 6 to 12 h, and with protein A/G-agarose (Oncogene Science Inc.) for 4 h, then subjected to DNA binding assay. The reaction mixture was inverted gently for 6–12 h at 4 °C. DNA-cellulose was recovered by centrifugation, washed four times with sufficient amounts of RIPA buffer, and DNA binding proteins were dissolved in Laemmli's sample buffer (31Suzuki K. Kodama S. Watanabe M. Radiat. Res. 1998; 149: 195-201Crossref PubMed Scopus (22) Google Scholar). Total cell extract was incubated with monoclonal antibodies against ATM or DNA-PK proteins for 12 and 6 h at 4 °C. Then Protein A/G-agarose was added and incubated for a further 12 and 6 h at 4 °C. The immunocomplexes were recovered by centrifugation at 15,000 rpm for 10 min at 4 °C. The supernatant was used as immunodepleted cell lysate. The resultant pellet was washed four times with RIPA buffer, dissolved in sample buffer, and electrophoresed on 5% SDS-polyacrylamide gel. The proteins were detected by Western blotting analysis as described above. Total cell extract containing closed circular pREP4 and Pvu II-digested was incubated with monoclonal antibodies against ATM protein for 6 h. Then protein A/G-agarose was added and incubated for further 6 h at 4 °C. The immunocomplexes were recovered by centrifugation at 15,000 rpm for 10 min at 4 °C. The resultant pellet was washed four times with RIPA buffer and resuspend in TE buffer (10 mm Tris-HCl, pH 7.2, 1 mm EDTA) containing 0.1 mg/ml proteinase K. The samples were incubated for 1 h at 50 °C, extracted with phenol/chloroform/isoamyl alcohol, and precipitated with ethanol. The plasmid DNA was resuspended in TE buffer and electrophoresed on 1% agarose gel. The anti-human ATM mouse monoclonal antibody used in this study was raised against human ATM amino acid sequence between 2577 and 3056. As shown in Fig. 1, the antibody recognized a single protein in normal human cells whose molecular mass was estimated to have approximately 370 kDa. The expression of the ATM protein was examined in two AT fibroblasts, AT2KY and AT5BI. These two cell lines have deletions in the ATM gene and these mutations are expected to cause ATM protein truncation. We found that no ATM protein was expressed in these two cell lines, while the immunoblotting using anti-DNA-PK antibody confirmed the equal amount of DNA-PK protein expressed in all three cells. The results indicate that the antibody detects ATM protein in normal human cells and that the mutations in two AT cell lines destabilize ATM protein. The level of ATM protein in normal human cells irradiated with 4 Gy of x-rays was examined by Western blotting (Fig. 2). The ATM protein was found in the nuclear proteins, and its amount did not change for up to 12 h after X-irradiation (Fig. 2, middle). The abundance of ATM protein in the cytoplasm also did not change (Fig. 2, top), indicating that ionizing radiation did not affect ATM protein stability or nuclear localization. In contrast, bi-phasic accumulation of the p53 protein was detected (Fig. 2, bottom). p53 protein levels increased 1 h after irradiation, transiently decreased to a control level, and increased again at 8 h after irradiation. We also confirmed that transient decreases in p53 protein between 4 and 6 h are due to increased expression of MDM2 protein induced by p53 (data not shown). In order to examine whether ATM protein recognizes damaged DNA, total cell lysate prepared from unirradiated normal human cells or cells incubated for 2 h after X-irradiation with 6 Gy was mixed with double strand DNA-cellulose, and the proteins associated with DNA-cellulose were subjected to Western blot analysis (Fig.3). ATM protein in lysate from unirradiated cells was found in the proteins bound to native double strand DNA (Fig. 3 A), and the amount of ATM protein did not change when cells were irradiated with 6 Gy of x-rays and incubated for 2 h, which caused the efficient accumulation of p53 protein (Fig.2). In contrast, the association of the ATM protein to double strand DNA was significantly increased by more than 3-fold when DNA-cellulose irradiated with 20 Gy of x-rays in vitro was used (Fig.3 A). In order to confirm whether the association was specific to DNA, cell lysate first incubated with unirradiated DNA-cellulose was mixed with irradiated DNA-cellulose (Fig.3 B). We found that the ATM protein was again recovered as the protein associated with irradiated DNA. Similarly, when cell lysate mixed with unirradiated DNA was then incubated with unirradiated DNA, ATM protein was recovered as DNA-associated protein, although there was little or no ATM protein recovered if the cell lysate was first incubated with irradiated DNA-cellulose. The results indicate that ATM protein specifically associates with double strand DNA and that ATM protein interacts more efficiently with damaged DNA. In order to clarify whether the enhanced interaction with irradiated DNA is dependent on DNA double strand breaks, cell lysate was mixed with double strand DNA-cellulose treated with either 5 kJ/m2 UVC or 1000 units of Pvu II for 4 h or with single strand DNA exposed to 20 Gy of x-rays (Fig.4 A). We found that increased association of ATM protein was observed only if the double strand DNA was treated with Pvu II, indicating that the ATM protein preferentially recognizes double strand breaks. In Fig. 4 B, the association of ATM protein with x-ray-irradiated double strand DNA was examined in a buffer containing different concentrations of NaCl. ATM association was not affected at NaCl concentrations between 150 and 450 mm, but it was significantly reduced at 550 mm NaCl. The interaction of ATM protein with damaged DNA was further examined by DNA coprecipitation assay (Fig.5). Closed circular and Pvu II-digested plasmid DNA was mixed with total cell extract, and then immunoprecipitates using anti-ATM antibody were subjected to agarose gel electrophoresis. As shown in Fig.5 B, the immunoprecipitation coprecipitated linear Pvu II-digested DNA, confirming the association of ATM protein with damaged double strand DNA.Figure 5DNA coprecipitation. Control cells were lysed in RIPA buffer (150 mm NaCl), and 400 μl of the extract (1 mg/ml) containing 5 μg of genomic DNA extracted from normal human (HE49) cells, 5 μg of closed circular pREP4 plasmid (Invitrogen, NV Leek, The Netherlands), and 5 μg of Pvu II-digested pREP4 was incubated with monoclonal antibody against ATM protein for 6 h at 4 °C. Then 40 μl of protein A/G-agarose (Oncogene Research Products, Cambridge, MA) was added, and incubated for further 6 h at 4 °C. The immunocomplexes were recovered by centrifugation at 15,000 rpm for 10 min at 4 °C. The resultant pellet was washed 4 times with RIPA buffer, and resuspend in TEN buffer (10 mm Tris-HCl, pH 7.2, 1 mm EDTA, 150 mm NaCl) containing 0.1 mg/ml proteinase K. The samples were incubated for 1 h at 50 °C, extracted twice with phenol/chloroform/isoamyl alcohol, and precipitated with ethanol. The plasmid DNA was resuspended in TE buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA), and electrophoresed on 1% agarose gel.A, the immunoprecipitates were subjected to SDS-PAGE and ATM protein was detected by Western blot analysis. Total cell extract (lane 1) was incubated in the absence (lane 2) or in the presence (lane 3) of ATM antibody. B, total cell extract containing both closed circular and Pvu II-digested plasmids (lane 1) was immunoprecipitated in the absence (lane 2) or in the presence (lane 3) of ATM antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The putative leucine zipper motif at codons 1217–1238 suggests that the ATM protein may form homodimers or heterodimers with other proteins. DNA-PK also has a leucine zipper motif, and it recognizes DNA double strand breaks by interaction with Ku proteins. Therefore, we examined whether ATM and DNA-PK form heterodimers to recognize damaged DNA. We found that immunoprecipitates using anti-ATM antibody contained small amounts of DNA-PK protein, while the antibody immunoprecipitated significant amounts of ATM protein (Fig. 6). Similarly, anti DNA-PK antibody immunoprecipitated large amounts of DNA-PK protein, whereas only small amounts of ATM protein was recovered. These results indicate that only a small fraction of ATM protein interacts with DNA-PK protein in vitro. In order to clarify whether DNA-PK protein is required for the association of ATM protein with damaged DNA, immunodepleted cell lysate was used for DNA binding assays. As shown in Fig. 7, ATM depletion significantly reduced the amount of ATM protein but not DNA-PK protein in cell lysate (Fig. 7A and 7B). Similarly, depletion of DNA-PK did not affect the amount of ATM protein. As shown in Fig. 7 C, ATM protein depletion significantly decreased the amount of ATM protein interacting with DNA, whereas DNA-PK depletion did not affect its association. Thus, it can be concluded that the association of ATM protein with damaged DNA does not required DNA-PK protein. Furthermore, cell lysates obtained from xrs-5 and xrs-6 cells were used in order to examine whether Ku proteins are involved in an association of the ATM protein with damaged DNA (Fig. 8). In xrs-5 cells, there were no mutations in the Ku80 gene; however, very low levels of Ku80 transcription were found (29Singleton B.K. Priestley A. Steingrimsdottir H. Gell D. Blunt T. Jackson S.P. Lehmann A.R. Jeggo P.A. Mol. Cell. Biol. 1997; 17: 1264-1273Crossref PubMed Scopus (165) Google Scholar). The Ku80 gene in xrs-6e cells carries an insertion that results in a frameshift and the resultant Ku80 protein was predicted to be a truncated protein (29Singleton B.K. Priestley A. Steingrimsdottir H. Gell D. Blunt T. Jackson S.P. Lehmann A.R. Jeggo P.A. Mol. Cell. Biol. 1997; 17: 1264-1273Crossref PubMed Scopus (165) Google Scholar). Immunoblotting analysis proved there was no detectable Ku80 protein in either cells (data not shown). As shown in Fig. 8, the antibody used in this study recognizes ATM protein in CHO, xrs-5 and xrs-6e cells, although the amount of ATM protein was slightly decreased in xrs-5 and xrs-6e cells. The cell lysates obtained from CHO, xrs-5, and xrs-6 cells were mixed with double strand DNA-cellulose irradiated with 20 Gy of x-rays. Although the amounts of ATM protein associated with damaged DNA were less in xrs-5 and xrs-6e cells than in CHO cells, the ATM protein in all of the cells could interact with damaged DNA.Figure 7Effect of immunodepletion of ATM and DNA-PK proteins on ATM association with damaged DNA. Total cell extracts obtained from control cells were incubated with antibodies against ATM (ATM-depleted) or DNA-PK (DNA-PK-depleted) for 12 and 6 h, respectively. The samples were then mixed with protein A/G-agarose, incubated for 6–12 h at 4 °C, and centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatant was screened for ATM and DNA-PK protein amounts (A and B, respectively), or it was subjected to a DNA binding assays (C). The expression levels of ATM (A and C) and DNA-PK (B) proteins were analyzed by Western blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Association of ATM protein with double strand DNA in Chinese hamster cells. Total cell extracts were obtained from CHO (A and B, lanes 1 and 4), xrs-5 (A and B, lanes 2 and 5), and xrs-6e (A and B, lanes 3 and 6) cells. Sixteen μg of samples were subjected to SDS-PAGE, and the proteins were analyzed by Western blot analysis (A and B, lanes 1–3). DNA binding assay was performed using native double-stranded DNA-cellulose irradiated with 20 Gy of x-rays. Cells were lysed in RIPA buffer containing 350 mm NaCl, and total cell extracts obtained from control cells were incubated with 20 Gy-irradiated DNA-cellulose (A and B, lanes 4–6) for 12 h at 4 °C, washed four times with sufficient amounts of RIPA buffer (350 mm NaCl), and the proteins bound to DNA-cellulose were analyzed by Western blot analysis. The proteins were detected using monoclonal antibodies against ATM (A) and DNA-PK (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The present study shows that approximately 350-kDa protein was detected as ATM protein in normal human embryo cells using anti-ATM monoclonal antibody used in this study (Fig. 1). We did not detect ATM protein in two AT cell lines, AT2KY and AT5BI. AT2KY cells have a deletion in the ATM gene at nucleotide position 7883, which is predicted to form a truncated ATM protein (32Gilad S. Khosravi R. Shkedy D. Uziel T. Ziv Y. Savitsky K. Rotman G. Smith S. Chessa L. Jorgensen T.J. Harnik R. Frydman M. Sanal O. Portnoi S. Goldwicz Z. Jaspers N.G. Gatti R.A. Lenoir G. Lavin M.F. Tatsumi K. Wegner R.D. Shiloh Y. Bar-Shira A. Hum. Mol. Genet. 1996; 5: 433-439Crossref PubMed Scopus (259) Google Scholar). AT5BI cells have a deletion of 6 base pairs in the ATM gene, which may cause deletion of two amino acids at codon 2427 (32Gilad S. Khosravi R. Shkedy D. Uziel T. Ziv Y. Savitsky K. Rotman G. Smith S. Chessa L. Jorgensen T.J. Harnik R. Frydman M. Sanal O. Portnoi S. Goldwicz Z. Jaspers N.G. Gatti R.A. Lenoir G. Lavin M.F. Tatsumi K. Wegner R.D. Shiloh Y. Bar-Shira A. Hum. Mol. Genet. 1996; 5: 433-439Crossref PubMed Scopus (259) Google Scholar). Because the antibody used in this study recognizes amino acids between 2577 and 3056, it is possible that the ATM protein in these AT cell lines will be destabilized, as suggested previously (25Brown K.D. Ziv Y. Sadanandan S.N. Chessa L. Collins F.S. Shiloh Y. Tangle D.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1840-1845Crossref PubMed Scopus (150) Google Scholar, 26Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Zhang N. Farrell A. Ramsey J. Gatti R. Lavin M. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (170) Google Scholar). The time course experiments showed that the amount of ATM protein in both nuclear and cytoplasm did not change up to 12 h after X-irradiation, while p53 accumulated significantly during this period. Previous studies have also shown that ionizing radiation and UV irradiation do not affect the amount of ATM protein (24Lakin N.D. Weber P. Stankovic T. Rottinghaus S.T. Taylor A.M.R. Jackson S.P. Oncogene. 1996; 13: 2707-2716PubMed Google Scholar, 25Brown K.D. Ziv Y. Sadanandan S.N. Chessa L. Collins F.S. Shiloh Y. Tangle D.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1840-1845Crossref PubMed Scopus (150) Google Scholar, 26Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Zhang N. Farrell A. Ramsey J. Gatti R. Lavin M. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (170) Google Scholar). Therefore, it can be hypothesized that ATM protein is not an inducible mediator or a transducer in signal transduction which activates p53 protein, but it acts as a sensor for DNA damage. Recently, it has been reported that ATM protein associates with chromatin in meiotic prophase (27Keegan K.S. Holtzman D.A. Plug A.W. Christenson E.R. Brainerd E.E. Flaggs G. Bentley N.J. Taylor E.M. Meyn M.S. Moss S.B. Carr A.M. Ashley T. Hoekstra M.F. Gene Dev. 1996; 10: 2423-2437Crossref PubMed Scopus (254) Google Scholar). Furthermore, in ATM−/− knockout mice, it has been shown that meiosis is arrested at prophase, which results in an abnormal chromosomal synapsis and chromosome fragmentation (33Xu Y. Ashley T. Brainerd E.E. Bronson R.T. Meyn M.S. Baltimore D. Genes Dev. 1996; 10: 2411-2422Crossref PubMed Scopus (748) Google Scholar). Because DNA double strand breaks are thought to occur in meiotic prophase, it has been hypothesized that ATM recognizes DNA strand breaks (27Keegan K.S. Holtzman D.A. Plug A.W. Christenson E.R. Brainerd E.E. Flaggs G. Bentley N.J. Taylor E.M. Meyn M.S. Moss S.B. Carr A.M. Ashley T. Hoekstra M.F. Gene Dev. 1996; 10: 2423-2437Crossref PubMed Scopus (254) Google Scholar, 33Xu Y. Ashley T. Brainerd E.E. Bronson R.T. Meyn M.S. Baltimore D. Genes Dev. 1996; 10: 2411-2422Crossref PubMed Scopus (748) Google Scholar). Therefore, we examined whether ATM protein associates with damaged DNA. As shown in Figs. 3 and 4, ATM protein was found to interact with double strand DNA, and its association markedly increased when DNA was exposed to ionizing radiation, indicating that ATM constitutively interacts with DNA, and it monitors DNA damage. We found that irradiation of cells did not alter the amount of ATM protein associated with damaged DNA. Thus, it seems likely that increased association with damaged DNA may not require posttranslational modification, although ATM protein is a phosphoprotein (34Chen G. Lee E.Y.-H. J. Biol. Chem. 1996; 271: 33693-33697Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The results presented in Fig. 4 demonstrate that the association of ATM protein with DNA was also increased using DNA-damaged by P vu II endonuclease treatment, but not by DNA exposed to UV light. X-irradiated denatured DNA was not a good substrate for ATM association. P vu II treatment produces blunt-ended double strand breaks, and UV light causes pyrimidine dimers and (6-4) photoproducts predominantly. Thus, it is implicated that ATM protein is recruited to the site of DNA double strand breaks and it functions as a sensor for DNA damage. The amino acid sequence of the ATM protein has a putative leucine zipper motif at codons 1217–1238 (6Savitsky K. Sfez S. Tagle D.A. Ziv Y. Sartiel A. Collins F.C. 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Since DNA-PK has been reported to have a putative leucine zipper motif at codons 1503–1538 (5Hartley K.O. Gell D. Smith G.C.M. Zhang H. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Anderson C.W. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (674) Google Scholar), we determined whether ATM protein associated with damaged DNA through interaction with DNA-PK. As shown in Fig. 6, the association between ATM and DNA-PK was very weak. Furthermore, depletion of DNA-PK did not affect the association of ATM with damaged DNA (Fig. 7). The association of ATM protein with damaged DNA was also detected in Chinese hamster cells deficient in Ku80 protein (Fig. 8). Therefore, both DNA-PK and Ku80 proteins are probably not involved in the recognition of damaged DNA by ATM protein. So far, DNA-PK and ATM have implicated to function independently (37Jongmans W. Artuso M. Vuillaume M. Bresil H. Jackson S.P. Hall J. Oncogene. 1996; 13: 1133-1138PubMed Google Scholar); however, recent reports have indicated that c-Abl protein interacts with both DNA-PK and ATM proteins (38Baskaran R. Wood L.D. Whitaker L.L. Canman C.E. Morgan S.E. Xu Y. Barlow C. Baltimore D. Wynshaw-Boris A. Kastan M.B. Wang J.Y.J. Nature. 1997; 387: 516-519Crossref PubMed Scopus (487) Google Scholar, 39Kharbanda S. Pandey P. Jin S. Inoue S. Bharti A. Yuan Z.-M. Weichselbaum R. Weaver D. Kufe D. Nature. 1997; 386: 732-735Crossref PubMed Scopus (239) Google Scholar, 40Shafman T. Khanna K.K. Kedar P. Spring K. Kozlov S. Yen T. Hobson K. Gatei M. Zhang N. Watters D. Egerton M. Shiloh Y. Kharbanda S. Kufe D. Lavin M.F. Nature. 1997; 387: 520-523Crossref PubMed Scopus (419) Google Scholar), and therefore, there is a possibility that these two independent pathways may communicate or cooperate to transduce signals. Further studies will be needed to clarify this point. In conclusion, we have shown that ATM protein associates with DNA in normal human cells, and the association is potentiated by DNA double strand breaks induced by ionizing radiation. The recruited ATM protein may be activated by an unknown mechanism and induce a signal to stabilize p53, thereby regulating cell cycle, DNA replication, apoptosis, and DNA repair. We thank T. K. Hei, W. F. Morgan, and P. A. Jeggo for materials that made this study feasible.