Proc. Nati. Acad. Sci. USA Vol. 91, pp. 6904-6908, July 1994 Biochemistry DNA-dependent protein kinase (Ku protein-p350 complex) assembles on double-stranded DNA (tnscription factor/hum autoant-gen Ku) AKIRA SUWA*t, MICHITO HIRAKATA*, YOSHIHIKo TAKEDA*, STEPHEN A. JESCH§, TSUNEYO MIMORIu, AND JOHN A. HARDIN*¶ *Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-3100; tDepartment of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan; and IDepartment of Chemistry and Biochemistry, Campus Box 205, University of Colorado, Boulder, CO 80309 Communicated by Roger D. Kornberg, March 1, 1994 (receivedfor review July 20, 1993)
AMBSTRACT The Ku protein Is an autoantigen that c si purified components are recombined in the presence ofDNA. of 70- and 80-kDa polypeptides. It a ates with double- We conclude that the DNA-PK holoenzyme is a stable stranded DNA at free ends. In the present study, we examined complex of Ku protein and p350 that forms on DNA. the ability of anti-Ku antibodies to hnmupr tate various structures from extracts ofHeLa cells prepared at different salt MATERIALS AND METHODS concentrations. Under physiological conditions, these antibod- ies identified a complex coinin the Ku protein and the AntiSera. Patient anti-sera containing anti-Ku antibodies 350-kDa component (p350) of DNA-dependent protein kinase were obtained from Japanese patients with various rheumatic (DNA-PK), which appeared to be dosely asscited on the diseases (1, 5). Mouse monoclonal antibodies specific forp80 DNA strand. In reconstitution experiments with cell extracts (mAb 111) were a gift from Westley H. Reeves (University of and biochemically purified components, the Ku protein-Sp30 North Carolina) (3>. Anti-p350 rabbit polyclonal antibodies complex formed only in the presence ofdouble-stranded DNA. (serum 9543-2) were a gift from Stephen P. Jackson (Well- The reconstituted complex was catalytically active. Together come/CRC Institute). Anti-RNAP II antibodies (Promega) with previous studies, these results indicate that the Ku protein were mouse monoclonal antibodies that recognize the CTDof interacts with DNA to create a binding site for p350 as the RNAP II. Anti-DNA anti-serum was from a patient with DNA-PK holoenzyme assembles. systemic lupus erythematosus. This serum gave a strong positive result in the Farr assay but immunoprecipitated no It has recently become apparent that the Ku protein is proteins (including histones) from [35S]methionine-labeled functionally related to a 350-kDa polypeptide (p350) associ- cell extracts. A mouse serum to p350 was prepared by ated with an enzymatic activity known as DNA-dependent immunizing BALB/c mice with biochemically purified p350. protein kinase (DNA-PK) (1-15) or template-associated pro- Preparation of DNA. Restriction fragments of double- tein kinase (16). This enzyme is an abundant nuclear com- stranded DNA were prepared by digestion of plasmid ponent that phosphorylates a number oftranscription factors pBR322 DNA (4361 bp; New England Biolabs) with Hae III as well as the C-terminal domain (CTD) ofthe largest subunit (Boehringer Mannheim), which recognizes 22 restriction ofRNA polymerase II (RNAP II) (17-20). In this capacity, it sites within this DNA. Single-stranded DNA was prepared is thought to play a key role in initiation of transcription from bacteriophage M13mpl8. (21-24). Radiolabeled Cell Extracts. HeLa cells were labeled with Active preparations of DNA-PK that have been purified [35S]methionine (Amersham), and cell extracts were pre- biochemically contain p350 along with the p70 and p80 Ku pared as described (5). Immunoprecipitation (IPP) buffer was subunits (25, 26). Moreover, recent studies by Dvir et al. (16) used for cell extractions and consisted of10 mM Tris HC1, pH and Gottlieb and Jackson (15) have demonstrated that bio- 7.5/0.1% Nonidet P-40/0.5 mM phenylmethylsulfonyl fluo- chemically purified p350 is catalytically active only in the ride/i pg of leupeptin per ml/i ug of aprotinin per ml/ presence ofthe Ku protein and that anti-Ku antibodies inhibit variable amounts ofNaCl (ranging in 0.05-M increments from active DNA-PK preparations. In UV light crosslinking ex- 0.05 to 0.5 M NaCl). These buffer conditions were used for periments, Ku protein mediated direct interaction of p350 all of the cell extractions described below. In some experi- with DNA (15). In gel mobility-shift assays, p350 produced a ments, cell extracts were incubated with EtdBr (50 pg/ml) at supershift of DNA-Ku protein complexes but when tested 40C for 30 min. alone did not alter mobility of free DNA (16). These studies Reassembly Experiments. Cell extracts prepared with 0.5 M led to the conclusion that Ku protein regulates binding of IPP buffer were dialyzed into 0.15 M IPP buffer at 40C for 8 p350 to DNA. It remained to be determined whether this hr prior to immunoprecipitation. In some experiments, cell process involved a stable interaction ofKu protein with p350, extracts were digested with DNase I (150 units per 2 x 106 occurred via an intervening DNA segment, or involved a cells; Pharmacia) at 37C for 30 min. To remove residual transient interaction of Ku protein with either DNA or p350 DNA fragments, cell extracts were incubated with the silica that triggered the latter to bind DNA. II In the present studies, we demonstrate an immunoprecip- matrix ofGeneclean and Mermaid kits (BIO 101) at 40C for itable Ku protein-p350 complex. This complex assembles in 10 min. In add-back experiments, DNA was restored with the presence of double-stranded DNA, is stable in physio- addition of 300 ng of Hae III-digested pBR322 double- logic buffers, is resistant to DNase digestion and the presence stranded DNA or M13mpi8 single-stranded DNA. After of EtdBr, and can be reconstituted when its separately Abbreviations: DNA-PK, DNA-dependent protein kinase; CTD, C-terminal domain; RNAP II, RNA polymerase II. The publication costs ofthis article were defrayed in part by page charge tPresent address: Tokyo Metropolitan Ohtsuka Hospital, 2-8-1 Mi- payment. This article must therefore be hereby marked "advertisement" namiohtsuka, Toshima-ku, Tokyo 170, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 6904 Downloaded by guest on September 29, 2021 Biochemistry: Suwa et aL Proc. NatL. Acad. Sci. USA 91 (1994) 6905 addition ofthese DNAs, cell extracts were maintained at 4TC by the corresponding antiserum in immunoblots. Also, we for 30 min, and immunoprecipitation was carried out imme- observed that V8 protease digestion products (31) were diately thereafter to minimize any further DNase I activity. identical for the 350-kDa polypeptides that immunoprecipi- Ku protein and p350 were isolated and used in kinase tate with either anti-p350 or anti-Ku antibodies (data not assays as described (20, 27). Immunoblot and immunopre- shown). The rabbit anti-p350 antiserum did not coimmuno- cipitation assays were performed according to standard pro- precipitate the Ku polypeptides, possibly because it binds tocols (28-30) with bound antibodies detected enzymatically. epitopes that are accessible only after denaturation orthat are In some immunoprecipitation experiments, IgG was linked blocked in the Ku protein-p350 complex. covalently to Sepharose beads with dimethyl pimelimidate. As shown in Fig. 2, six different anti-Ku anti-sera immu- noprecipitated a polypeptide of 350 kDa along with the Ku RESULTS polypeptides when tested under conditions of 0.15 M NaCl. Inununoprecipitation ofKu Protehn-p350 Complexes. Initial In contrast, antibodies to a variety of other nuclear autoan- studies were carried out with IPP buffer. A broad array of tigens and a normal human serum did not immunoprecipitate nonspecifically incorporated proteins appeared in anti-Ku the Ku protein-p350 complex. The exception is anti-DNA antibody-mediated immunoprecipitates prepared at 0.05 M antibodies used in lane 8. Here it can be seen that p70, p80, NaCl. In contrast, immunoprecipitates prepared at a salt and p350 are included along with an array of other polypep- concentration > 0.3 M NaCl contained little other than the tides, presumably because they coimmunoprecipitate with p70 and p80 Ku heterodimer. Thus, in subsequent experi- the DNA fragments that are solubilized as cell extracts are ments, cell extractions and immunoprecipitations were per- prepared. formed with buffers containing 0.15 M NaCl (physiologic salt) The Ku Protein-p350 Complex Assembles in Vitro in the or 0.5 M NaCl (high 'salt), which is a standard immunopre- Presence of DNA. To explore the salt sensitivity of Ku cipitation condition (5, 28, 29). protein-p350 complexes, we prepared cell extracts under As shown in Fig. 1, anti-p350 antibodies immunoprecipi- conditions of 0.5 M NaCl and subsequently dialyzed them tated a single polypeptide of %350 kDa under conditions of into 0.15 M IPP buffer. As shown in Fig. 3, immunoprecip- 0.15 M and 0.5 M NaCl (Fig. 1A). At physiologic salt itations were carried out periodically with anti-Ku and anti- conditions, various anti-Ku antibodies immunoprecipitated DNA antibodies. It can be seen that p70 and p80 were readily p70 and p80 as well as the 350-kDa polypeptide (lanes 3 and immunoprecipitated at each time point (Fig. 3A). However, 5). At 0.5 M NaCi, immunoprecipitates contained only the after 8 hr of dialysis the immunoprecipitates incorporated Ku components (lanes 4 and 6). The absence of the 350-kDa p350, indicating that Ku protein-p350 complexes reassemble band in the latter experiments argues strongly that none ofthe as physiologic salt conditions are approached. Immunopre- sera contains antibodies that bind this polypeptide directly. cipitations were also carried out with anti-DNA antibodies to The 350-kDa polypeptide is identified as the catalytic com- confirm that these proteins were associating with DNA. As ponent (p350) ofDNA-PK in Fig. 1B because it is recognized shown in Fig. 3A Right, anti-DNA antibodies immunopre- cm~
Lo I- 0 A CYI C/) ClD C:L CoIL IC CX5 C6 e75 I;Ct B
CM)0) CC)
C2; - Ct -_ ZC_} - CD - x C2Z- CalX zCal CC Cd)2 xX C)}X L) a. X lr z . i n L n Xr T- M C-) CZ U) CO ) CD CD CD E) C f° LO C7 C)~ CD CD CCD & C kDa -p350 kDa p)35 200- 200 --
97.4- 97.4-- - p80 p80 69- * ;i- n 69 - -- p70
46 - 46 30- 30 -
FIG. 1. Anti-Ku antibodies immunoprecipitate a dissociable complex ofKu protein and p350. (A) Autoradiogram demonstrating polypeptides immunoprecipitated under different salt conditions. mAb 111 is a mouse monoclonal antibody that recognizes p80. Anti-Ku antiserum OM also contains anti-Ro antibodies. (B) Identification of the 350-kDa polypeptide as the p350 component of DNA-PK. Immunoprecipitates were prepared with serum OM at 0.15 M NaCl and used as substrate in immunoblots. Individual strips were probed with antibodies as indicated. NHS, normal human serum; NRS, normal rabbit serum. Downloaded by guest on September 29, 2021 6906 Biochemistry: Suwa et aL Proc. NatL. Acad. Sci. USA 91 (1994) To assess the extent of DNA cleavage in these experi- CN) ments, immunoprecipitation was also carried out using anti- Ln .__ 1-- r .- c 0 DNA antibodies. Again as in Fig. 3A, immune complexes C) t E~] 0 a from undigested or lightly digested cell extracts contained a CO = = = a _ (
97.4 699.ofHi 97.4- 1 _~~~ Ha -~dB* -p80p70 -p80 69- X -- p70 46- 46-
30 -
FIG. 3. Stability of the Ku protein-p350 complex. (A) [35SJMethionine-labeled HeLa cell extracts were prepared with 0.5 M IPP buffer and dialyzed into 0.15 M IPP buffer. Aliquots were used for immunoprecipitation at different time intervals. (B) Extracts of [35S]methionine-labeled HeLa cells were digested with 15 or 150 units of DNase I per 2 x 106 cells or incubated with EtdBr and subsequently immunoprecipitated with either anti-Ku or anti-DNA antibodies. Downloaded by guest on September 29, 2021 Biochemistry: Suwa et al. Proc. Natl. Acad. Sci. USA 91 (1994) 6907 kDa A i L - p350 200- ;a c: kDa
97.4- -- P350 _____.unowe - -p80 200- 69- Ad *- GC14To *- GO147a 46- 97.4 - -p80 30- 69 - -- p70 ,~ _ wo_ _mb_
1 2 3 4 5 46 - FIG. 4. The Ku protein-p350 complex assembles on double- stranded DNA. [35S]Methionine-labeled HeLa cells were extracted with 0.5 M IPP buffer, treated to remove DNA, and dialyzed into 0.15 B DNAAUUAS M IPP buffer. Immunoprecipitation was carried out with anti-Ku Ku antiserum OM. Lanes: 1, immunoprecipitate prepared from 0.5 M NaC1 cell extract after DNase I digestion (150 units per 2 x 106 cells; CD~~~~~~~0 37TC for 30 min); 2, immunoprecipitate after adjusting salt content to 0.15 M NaCl; 3, immunoprecipitate after complete removal of small =6 CZ CZ 1C CZ DNA fragments with silica gels; 4, immunoprecipitate after addition kDa of double-stranded DNA (600 ng/ml; subsequent incubation at 4TC - p350 for 30 min); 5, immunoprecipitate after addition of single-stranded DNA (same incubation as lane 4). 200 - various antibodies using 0.15 M IPP buffer. It is shown in Fig. SA that catalytically active DNA-PK assembled in this t - GC147o reaction mixture and could be immunoprecipitated with - GC147a 97.4 - antibodies to Ku protein, DNA, or the CTD of RNAP II but not with normal human serum. As expected from previous studies, the GC147 target is heavily phosphorylated, and the 69 - Ku polypeptides (p70 >> p80) and p350 are autophosphory- lated (27). The necessity ofindividual factors for formation of an active DNA-PK complex is shown in Fig. SB. Assembly components were combined in the presence of[y-32P]ATP, or FIG. 5. Immunoprecipitable Ku protein-p350 complexes are DNA and Ku protein were separately omitted. Antibodies to catalytically active. (A) Biochemically purified Ku protein, p350, the were Ku, p350, and RNAP II immunoprecipitated the GC147o target protein GC147, and the DNA template AdUAS incubated phosphorylation target when all components of the complex together in the presence of ('y-32P]ATP and immunoprecipitated with various antibodies as shown. GC147o is the hyperphosphorylated were combined. In the absence of either Ku protein or DNA, form ofGC147a. The latter is visualized because it is phosphorylited catalytic activity was either minimal or absent. These results at only a few sites. (B) All constituents were combined (lanes 1-3) or demonstrate that catalytically active complexes of DNA-PK Ku protein (lanes 4-6) and DNA (lanes 7-9) were omitted as shown. are sufficiently stable to undergo immunoprecipitation, and Immunoprecipitations were performed with the indicated antibodies. they suggest that the complexes detected in HeLa cell The anti-p350 antiserum was a polyclonal mouse serum prepared in extracts are also catalytically active. our laboratory. This serum is monospecific for p350 in immunopre- cipitations carried out at 0.5 M NaCl and coprecipitates Ku protein in studies performed at 0.15 M NaCl. DISCUSSION The present observations extend recent insights into the complex was not observed in earlier immunoprecipitation biological function of the Ku protein through the demonstra- experiments because ofits instability in the buffers ordinarily tion that this protein assembles into a catalytically active used for these studies (5, 28). The high level of solubility of complex with p350 in the presence of linear double-stranded the Ku protein-p350-DNA structures is likely to reflect their DNA. This is the DNA form that activates DNA-PK and release from intact chromatin as DNA is sheared in the interacts with Ku protein, initially at its free ends (9, 25, 26). course of cell sonification. Indeed, such cell extracts may be These results are consistent with earlier observations ofDvir enriched in transcriptionally active chromatin fiagments et al. (16) showing that p350 induces a supershift in Ku since the relatively open chromatin structure within such protein-DNA gel mobility and those of Gottlieb and Jackson regions may enhance susceptibility to fragmentation and (15) demonstrating the presence of Ku protein and p350 in solubilization. some immunoprecipitates prepared with anti-Ku antibodies. Several arguments indicate that Ku protein and p350 are The DNA-PK complex is highly sensitive to the ionic closely approximated on the DNA strand. The previous strength of the environment, as are many other structures studies demonstrated that UV light crosslinks p350 to.DNA that involve DNA-protein interactions. This complex was only in the presence of Ku protein (15). The present studies stable at 0.15 M NaCI but tended to dissociate progressively demonstrated that the Ku protein-p350 complex resists ex- as the salt concentration was increased from 0.2 to 0.3 M tended DNase I digestion and exposure to EtdBr, an agent NaCl. In the previous studies, we noted that Ku protein that disrupts mainly DNA-protein interaction. These latter dissociates completely from DNA in buffers containing 0.35 studies imply either that Ku protein and p350 are physically M NaCl (9). Thus, the interaction between p350 and Ku associated even after dissociation from the DNA strand or protein-DNA binding site may be somewhat less stable than that very small DNA figments serve to stabilize this com- the interaction of Ku protein with DNA. Most likely, this plex. We would add the caveat that we cannot exclude the Downloaded by guest on September 29, 2021 6908 Biochemistry: Suwa et al. Proc. Natl. Acad. Sci. USA 91 (1994) possibility that both Ku protein and p350 have unusual ability 6. Reeves, W. H. & Sthoeger, Z. M. (1989) J. Biol. Chem. 264, to bind to DNA that has intercalated EtdBr. These observa- 5047-5052. tion support a model in which Ku protein associates with an 7. Mimori, T., Ohosone, Y., Hama, N., Suwa, A., Akizuki, M., appropriate DNA domain to create a binding site for a p350 Homma, M., Griffith, A. J. & Hardin, J. A. (1990) Proc. Nati. molecule. Acad. Sci. USA 87, 1777-1781. 8. Griffith, A. J., Blier, P. R., Mimori, T. & Hardin, J. A. (1992) An important question now is, how does the Ku protein J. Biol. Chem. 267, 331-338. recognize a DNA site that is poised for transcription? In 9. Mimori, T. & Hardin, J. A. (1986) J. Biol. Chem. 261, 10375- earlier studies, we noted that Ku protein binds well to linear 10379. double-stranded DNA (9), which is the DNA form required 10. de Vries, E., van Driel, W., Bergsma, W. G., Arnberg, A. C. for DNA-PK activity in vitro (25, 26). However, it does not & van der Vliet, P. C. (1989) J. Mol. Biol. 208, 65-78. seem likely that free DNA ends regularly occur at transcrip- 11. Stuiver, M. H., Coenjaerts, F. E. J. & van der VTet, P. C. tion sites. Some evidence suggests that the Ku protein can (1990) J. Exp. Med. 172, 1049-1054. slide along the DNA strand, and it is possible that it associ- 12. Blier, P. R., Griffith, A. J., Craft, J. & Hardin, J. A. (1993) J. ates with DNA at some distal site and traverses along the Biol. Chem. 268, 7594-7601. 13. Paillard, S. & Strauss, F. (1991) Nucleic Acids Res. 19, 5619- DNA until an initiation complex is encountered (10). That 5624. concept would pose the problem of how Ku protein might 14. Jackson, S. P., MacDonald, J. J., Lees-Miller, S. & Tjian, R. traverse a DNA region bearing at least some nucleosomal (1990) Cell 63, 155-165. configurations. It should also be noted that the Ku protein 15. Gottlieb, T. M. & Jackson, S. P. (1993) Cell 72, 1-20. recognizes DNA forms other than those with free ends. For 16. Dvir, A., Peterson, S. R., Knuth, M. W., Lu, H. & Dynan, example, we have recently found that Ku protein binds well W. S. (1992) Proc. Nadl. Acad. Sci. USA 89, 11920-11924. to DNA nick sites (12). However, nicked circular DNA is not 17. Walker, A. I., Hunt, T., Jackson, R. J. & Anderson, C. W. a good activator of DNA-PK activity (25, 26). It is possible (1985) EMBO J. 4, 139-145. Ku bound to such sites interact with 18. Carter, T. H., Kopman, C. R. & James, C. B. L. (1988) Bio- that protein might chem. Biophys. Res. Commun. 157, 535-540. proteins other than p350 such as DNA repair components. 19. Lees-Miller, S. P. & Anderson, C. W. (1991) Cancer Cells 3, Binding of Ku protein to DNA hairpin loops has also been 341-346. noted (13), but again it is not clear that such DNA structures 20. Dvir, A., Stein, L. Y., Calore, B. L. & Dynan, W. S. (1993) J. relate to activation of transcription. Most recently, this Biol. Chem. 268, 10440-10447. protein was noted to bind to DNA minicircles bearing mis- 21. Cadena, D. L. & Dahmus, M. E. (1987) J. Biol. Chem. 262, matched (bubble) segments of 30 bp and to prefer DNA ends 12468-12474. bearing A-T rather than G-C termini, suggesting that it 22. Payne, J. M., Laybourn, P. J. & Dahmus, M. E. (1989) J. Biol. recognizes single- to double-stranded DNA transition sites in Chem. 264, 19621-19629. or bubbles it 23. Laybourn, P. J. & Dahmus, M. E. (1990) J. Biol. Chem. 265, promoter regions transcription (35). Thus, 13165-13173. appears that Ku protein utilizes its ability to recognize 24. Arias, J. A., Peterson, S. R. & Dynan, W. S. (1991) J. Biol. specific DNA forms to localize p350 to sites of active gene Chem. 266, 8055-8061. expression. 25. Carter, T. H., Vancurova, I., Sun, I., Lou, W. & DeLon, S. (1990) Mol. Cell. Biol. 10, 6460-6471. We thank Drs. William S. Dynan and Arik Dvir for helpful advice 26. Lees-Miller, S. P., Chen, Y.-R. & Anderson, C. W. (1990)Mol. and discussions, Dr. Westley H. Reeves for monoclonal antibodies Cell. Biol. 10, 6472-6481. to the Ku protein, and Dr. Stephen P. Jackson for rabbit polyclonal 27. Peterson, S. R., Dvir, A., Anderson, C. W. & Dynan, W. S. antibodies to p350. This work was supported by National Institutes (1992) Genes Dev. 6, 426-438. of Health Grants AR32549 (to J.A.H.) and GM35866 (to W. S. 28. Lerner, M. R. & Steitz, J. A. (1979) Proc. Nadl. Acad. Sci. Dynan, University of Colorado), a Biomedical Research Grant from USA 76, 5495-5499. the Arthritis Foundation, funds from the Georgia Research Alliance, 29. Mimori, T., Hinterberger, M., Petterson, I. & Steitz, J. A. and a generous donation from the Matuzak family. (1984) J. Biol. Chem. 259, 560-565. 30. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Nadl. 1. Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S. Acad. Sci. USA 76, 4350-4354. & Homma, M. (1981) J. Clin. Invest. 68, 611-620. 31. Cleveland, D. W., Fisher, S. G., Kirschner, M. W. & Laem- 2. Zweig, S. E., Rubin, S., Yaneva, M. & Busch, H. (1984) Proc. mli, U. K. (1977) J. Biol. Chem. 252, 1102-1106. Am. Assoc. Cancer Res. 25, 248 (abstr.). 32. Lai, J.-S. & Herr, W. (1992) Proc. Natl. Acad. Sci. USA 89, 3. Reeves, W. H. (1985) J. Exp. Med. 161, 18-39. 6958-6962. 4. Yaneva, M., Ochs, R., McRorie, D. K., Zweig, S. & Busch, H. 33. Lerman, L. S. (1961) J. Mol. Biol. 3, 18-30. (1985) Biochim. Biophys. Acta 841, 22-29. 34. Waring, M. (1970) J. Mol. Biol. 54, 247-279. 5. Mimori, T., Hardin, J. A. & Steitz, J. A. (1986) J. Biol. Chem. 35. Falzon, M., Fewell, J. W. & Kuff, E. L. (1993) J. Biol. Chem. 261, 2274-2278. 268, 10546-10552. Downloaded by guest on September 29, 2021