Proc. Nati. Acad. Sci. USAVol. 91, pp. 6904-6908, July 1994Biochemistry

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 UniversitySchool of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan; and IDepartment of Chemistry and Biochemistry, Campus Box 205, University ofColorado, Boulder, CO 80309

Communicated by Roger D. Kornberg, March 1, 1994 (received for review July 20, 1993)

AMBSTRACT The Ku protein Is an autoantigen that c siof 70- and 80-kDa polypeptides. It a ates with double-stranded DNA at free ends. In the present study, we examinedthe ability of anti-Ku antibodies to hnmupr tate variousstructures from extracts ofHeLa cells prepared at different saltconcentrations. Under physiological conditions, these antibod-ies identified a complex coinin the Ku protein and the350-kDa component (p350) of DNA-dependent protein kinase(DNA-PK), which appeared to be dosely asscited on theDNA strand. In reconstitution experiments with cell extractsand biochemically purified components, the Ku protein-Sp30complex formed only in the presence of double-stranded DNA.The reconstituted complex was catalytically active. Togetherwith previous studies, these results indicate that the Ku proteininteracts with DNA to create a binding site for p350 as theDNA-PK holoenzyme assembles.

It has recently become apparent that the Ku protein isfunctionally related to a 350-kDa polypeptide (p350) associ-ated with an enzymatic activity known as DNA-dependentprotein kinase (DNA-PK) (1-15) or template-associated pro-tein kinase (16). This enzyme is an abundant nuclear com-ponent that phosphorylates a number of transcription factorsas well as the C-terminal domain (CTD) of the largest subunitofRNA polymerase II (RNAP II) (17-20). In this capacity, itis thought to play a key role in initiation of transcription(21-24).

Active preparations of DNA-PK that have been purifiedbiochemically contain p350 along with the p70 and p80 Kusubunits (25, 26). Moreover, recent studies by Dvir et al. (16)and Gottlieb and Jackson (15) have demonstrated that bio-chemically purified p350 is catalytically active only in thepresence ofthe Ku protein and that anti-Ku antibodies inhibitactive DNA-PK preparations. In UV light crosslinking ex-periments, Ku protein mediated direct interaction of p350with DNA (15). In gel mobility-shift assays, p350 produced asupershift of DNA-Ku protein complexes but when testedalone did not alter mobility of free DNA (16). These studiesled to the conclusion that Ku protein regulates binding ofp350 to DNA. It remained to be determined whether thisprocess involved a stable interaction ofKu protein with p350,occurred via an intervening DNA segment, or involved atransient interaction of Ku protein with either DNA or p350that triggered the latter to bind DNA.

In the present studies, we demonstrate an immunoprecip-itable Ku protein-p350 complex. This complex assembles inthe presence of double-stranded DNA, is stable in physio-logic buffers, is resistant to DNase digestion and the presenceof EtdBr, and can be reconstituted when its separately

purified components are recombined in the presence ofDNA.We conclude that the DNA-PK holoenzyme is a stablecomplex of Ku protein and p350 that forms on DNA.

MATERIALS AND METHODSAntiSera. Patient anti-sera containing anti-Ku antibodies

were obtained from Japanese patients with various rheumaticdiseases (1, 5). Mouse monoclonal antibodies specific for p80(mAb 111) were a gift from Westley H. Reeves (University ofNorth Carolina) (3>. Anti-p350 rabbit polyclonal antibodies(serum 9543-2) were a gift from Stephen P. Jackson (Well-come/CRC Institute). Anti-RNAP II antibodies (Promega)were mouse monoclonal antibodies that recognize theCTD ofRNAP II. Anti-DNA anti-serum was from a patient withsystemic lupus erythematosus. This serum gave a strongpositive result in the Farr assay but immunoprecipitated noproteins (including histones) from [35S]methionine-labeledcell extracts. A mouse serum to p350 was prepared byimmunizing BALB/c mice with biochemically purified p350.

Preparation of DNA. Restriction fragments of double-stranded DNA were prepared by digestion of plasmidpBR322 DNA (4361 bp; New England Biolabs) with Hae III(Boehringer Mannheim), which recognizes 22 restrictionsites within this DNA. Single-stranded DNA was preparedfrom bacteriophage M13mpl8.

Radiolabeled Cell Extracts. HeLa cells were labeled with[35S]methionine (Amersham), and cell extracts were pre-pared as described (5). Immunoprecipitation (IPP) buffer wasused for cell extractions and consisted of10mM Tris HC1, pH7.5/0.1% Nonidet P-40/0.5 mM phenylmethylsulfonyl fluo-ride/i pg of leupeptin per ml/i ug of aprotinin per ml/variable amounts ofNaCl (ranging in 0.05-M increments from0.05 to 0.5 M NaCl). These buffer conditions were used forall of the cell extractions described below. In some experi-ments, cell extracts were incubated with EtdBr (50 pg/ml) at40C for 30 min.Reassembly Experiments. Cell extracts prepared with 0.5M

IPP buffer were dialyzed into 0.15 M IPP buffer at 40C for 8hr prior to immunoprecipitation. In some experiments, cellextracts were digested with DNase I (150 units per 2 x 106cells; Pharmacia) at 37C for 30 min. To remove residualDNA fragments, cell extracts were incubated with the silicamatrix ofGeneclean II and Mermaid kits (BIO 101) at 40C for10 min. In add-back experiments, DNA was restored withaddition of 300 ng of Hae III-digested pBR322 double-stranded DNA or M13mpi8 single-stranded DNA. After

Abbreviations: DNA-PK, DNA-dependent protein kinase; CTD,C-terminal domain; RNAP II, RNA polymerase II.tPresent address: Tokyo Metropolitan Ohtsuka Hospital, 2-8-1 Mi-namiohtsuka, Toshima-ku, Tokyo 170, Japan.ITo whom reprint requests should be addressed.

6904

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. NatL. Acad. Sci. USA 91 (1994) 6905

addition ofthese DNAs, cell extracts were maintained at 4TCfor 30 min, and immunoprecipitation was carried out imme-diately thereafter to minimize any further DNase I activity.Ku protein and p350 were isolated and used in kinase

assays as described (20, 27). Immunoblot and immunopre-cipitation assays were performed according to standard pro-tocols (28-30) with bound antibodies detected enzymatically.In some immunoprecipitation experiments, IgG was linkedcovalently to Sepharose beads with dimethyl pimelimidate.

RESULTSInununoprecipitation ofKu Protehn-p350 Complexes. Initial

studies were carried out with IPP buffer. A broad array ofnonspecifically incorporated proteins appeared in anti-Kuantibody-mediated immunoprecipitates prepared at 0.05 MNaCl. In contrast, immunoprecipitates prepared at a saltconcentration > 0.3 M NaCl contained little other than thep70 and p80 Ku heterodimer. Thus, in subsequent experi-ments, cell extractions and immunoprecipitations were per-formed with buffers containing 0.15M NaCl (physiologic salt)or 0.5 M NaCl (high 'salt), which is a standard immunopre-cipitation condition (5, 28, 29).As shown in Fig. 1, anti-p350 antibodies immunoprecipi-

tated a single polypeptide of %350 kDa under conditions of0.15 M and 0.5 M NaCl (Fig. 1A). At physiologic saltconditions, various anti-Ku antibodies immunoprecipitatedp70 and p80 as well as the 350-kDa polypeptide (lanes 3 and5). At 0.5 M NaCi, immunoprecipitates contained only theKu components (lanes 4 and 6). The absence of the 350-kDaband in the latter experiments argues strongly that none ofthesera contains antibodies that bind this polypeptide directly.The 350-kDa polypeptide is identified as the catalytic com-ponent (p350) ofDNA-PK in Fig. 1B because it is recognized

cm~Lo

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CX5

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0

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by the corresponding antiserum in immunoblots. Also, weobserved that V8 protease digestion products (31) wereidentical for the 350-kDa polypeptides that immunoprecipi-tate with either anti-p350 or anti-Ku antibodies (data notshown). The rabbit anti-p350 antiserum did not coimmuno-precipitate the Ku polypeptides, possibly because it bindsepitopes that are accessible only after denaturation or that areblocked in the Ku protein-p350 complex.As shown in Fig. 2, six different anti-Ku anti-sera immu-

noprecipitated a polypeptide of 350 kDa along with the Kupolypeptides when tested under conditions of 0.15 M NaCl.In contrast, antibodies to a variety of other nuclear autoan-tigens and a normal human serum did not immunoprecipitatethe Ku protein-p350 complex. The exception is anti-DNAantibodies used in lane 8. Here it can be seen that p70, p80,and p350 are included along with an array of other polypep-tides, presumably because they coimmunoprecipitate withthe DNA fragments that are solubilized as cell extracts areprepared.The Ku Protein-p350 Complex Assembles in Vitro in the

Presence of DNA. To explore the salt sensitivity of Kuprotein-p350 complexes, we prepared cell extracts underconditions of 0.5 M NaCl and subsequently dialyzed theminto 0.15 M IPP buffer. As shown in Fig. 3, immunoprecip-itations were carried out periodically with anti-Ku and anti-DNA antibodies. It can be seen that p70 and p80 were readilyimmunoprecipitated at each time point (Fig. 3A). However,after 8 hr of dialysis the immunoprecipitates incorporatedp350, indicating that Ku protein-p350 complexes reassembleas physiologic salt conditions are approached. Immunopre-cipitations were also carried out with anti-DNA antibodies toconfirm that these proteins were associating with DNA. Asshown in Fig. 3A Right, anti-DNA antibodies immunopre-

C/) ClD

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a. X lr z . i n L n Xr

) CD CD CD E) C f°M C-) CZ U)

C7 C)~ CD CD CDC & C

kDa

200-

97.4-

69 -

-p350 kDa

200 --

- p80-- p70

0)CM)

CC)

L)T-COLO

97.4--

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30-

30 -

FIG. 1. Anti-Ku antibodies immunoprecipitate a dissociable complex ofKu protein and p350. (A) Autoradiogram demonstrating polypeptidesimmunoprecipitated under different salt conditions. mAb 111 is a mouse monoclonal antibody that recognizes p80. Anti-Ku antiserum OM alsocontains anti-Ro antibodies. (B) Identification of the 350-kDa polypeptide as the p350 component of DNA-PK. Immunoprecipitates wereprepared with serumOM at 0.15M NaCl and used as substrate in immunoblots. Individual strips were probed with antibodies as indicated. NHS,normal human serum; NRS, normal rabbit serum.

Biochemistry: Suwa et aL

Proc. NatL. Acad. Sci. USA 91 (1994)

c 0

(<J0AV?2-) (2) aJO ru

__ _ _ SIK_ ., : . of.. ....... ... . S. .. .ha X-s w.sE ..., . . . A. i. .- s: i r

r M Aft JS,§. And

Hi USAHas..

91-" .

"'V-v -- W.---W.us

FIG. 2. Multiple anti-Ku antisera immunoprecipitate the Kuprotein-p350 complex. Cell extractions and immunoprecipitationswere carried out with 0.15 M IPP buffer and sera from variouspatients. Proteins were analyzed in a SDS 7.5% polyacrylamide gelfollowed by autoradiography. PCNA, proliferating cell nuclear an-tigen; NHS, normal human serum.

cipitated an array of proteins including small amounts of Kuprotein and p350.There remained the possibility that both Ku protein and

p350 might bind to DNA independently and be coimmuno-precipitated even though they do not physically interactthemselves. To resolve this issue, we examined how immu-noprecipitates are altered when cell extracts are treated withDNase I or EtdBr [which interacts with DNA in a relativelynonspecific manner to disrupt protein-DNA interactions(32)]. As shown in Fig. 3B, digestion with a small amount ofDNaseI increased the amount of immunoprecipitable Kuprotein-p350 complexes (lane 2), whereas more intense di-gestion led to a decrease (lane 3). Similarly, EtdBr diminishedbut did not abolish immunoprecipitable complexes (lane 4),suggesting that some Ku protein-p350 complexes remainintact in the absence of DNA. However, we cannot excludethe possibility that undetectable DNA fragments are respon-sible for maintaining complexes. It is also possible that Kuprotein and p350 differ from otherDNA binding proteins andremain associated with DNA, which has intercalated EtdBr(33, 34).

To assess the extent of DNA cleavage in these experi-ments, immunoprecipitation was also carried out using anti-DNA antibodies. Again as in Fig. 3A, immune complexesfrom undigested or lightly digested cell extracts contained anumber of polypeptides including the Ku subunits and p350(Fig. 3B, lanes 5 and 6). More intense DNase I digestion ortreatment with EtdBr prevented incorporation ofKu proteinand p350 into anti-DNA immunoprecipitates (lanes 7 and 8).From these experiments, we conclude that (i) DNase Iremoved DNA that could simultaneously associate withproteins and could be recognized by antibodies; (ii) EtdBrdestabilizes the Ku protein-p350 complex, possibly becauseKu protein does not bind toDNA that has intercalated EtdBr,and (iii) an apparently fragile complex of Ku protein-p350may persist after it exits from the DNA strand. In interpretingthese experiments, it should be recalled that DNA fragmentsas short as 12 bp activate DNA-PK activity (19). DNAfragments of this size may be produced by DNase I and couldaccount for the apparent increase in Ku protein-p350 com-plexes observed (Fig. 3B, lane 2). Such shortDNA fragmentsare not likely to be recognized well by anti-DNA antibodies.These experiments indicated that DNA serves to stabilize

the Ku protein-p350 complex and suggested that DNA mightbe required for its initial assembly. To test this possibility, weprepared cell extracts under 0.5 M NaCl salt conditions,digested them with DNase I, and adjusted salt conditions to0.15 M NaCl with dialysis. In some cases, extracts weretreated further with silica gels to remove any residual DNAfragments prior to dialysis into 0.15 M IPP buffer. As shownin Fig. 4, a Ku protein-p350 complex formed as the saltcontent of buffer was lowered (lanes 1 and 2). Formation ofthis complex appears to depend on the remaining presence ofsmall fragments ofDNA because it was not observed whenextracts were treated with silica matrix (lane 3), whichremoves DNA fragments of >10-bp. Subsequent addition ofdouble-stranded DNA (lane 4) promoted formation of thecomplex, but single-stranded DNA (ane 5) had no effect.The concern remained that the complexes detected in

HeLa cell extracts might not be analogous to active forms ofDNA-PK. To address this issue, Ku protein and p350 puri-fied biochemically from HeLa cells were combined withGC147 (GAL4-CTD fusion protein) and the AdUAS DNAtemplate (which contains GAL4 binding sites) in the presenceof [y32PJATP. Immunoprecipitations were performed with

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kDa

200- I

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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 anddialyzed into 0.15 M IPP buffer. Aliquots were used for immunoprecipitation at different time intervals. (B) Extracts of [35S]methionine-labeledHeLa cells were digested with 15 or 150 units of DNase I per 2 x 106 cells or incubated with EtdBr and subsequently immunoprecipitated witheither anti-Ku or anti-DNA antibodies.

CN)

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46 -

30 -

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(hrs)kDa

-- p350

-p80-- p70

6906 Biochemistry: Suwa et aL

Proc. Natl. Acad. Sci. USA 91 (1994)

kDa

200-- p350

97.4-_____.unowe - -p80

69- Ad

46-

30-,~ _ wo_ _mb_

1 2 3 4 5

FIG. 4. The Ku protein-p350 complex assembles on double-stranded DNA. [35S]Methionine-labeled HeLa cells were extractedwith 0.5M IPP buffer, treated to remove DNA, and dialyzed into 0.15M IPP buffer. Immunoprecipitation was carried out with anti-Kuantiserum OM. Lanes: 1, immunoprecipitate prepared from 0.5 MNaC1 cell extract after DNase I digestion (150 units per 2 x 106 cells;37TC for 30 min); 2, immunoprecipitate after adjusting salt content to0.15 M NaCl; 3, immunoprecipitate after complete removal of smallDNA fragments with silica gels; 4, immunoprecipitate after additionof double-stranded DNA (600 ng/ml; subsequent incubation at 4TCfor 30 min); 5, immunoprecipitate after addition of single-strandedDNA (same incubation as lane 4).

various antibodies using 0.15 M IPP buffer. It is shown in Fig.SA that catalytically active DNA-PK assembled in thisreaction mixture and could be immunoprecipitated withantibodies to Ku protein, DNA, or the CTD ofRNAP II butnot with normal human serum. As expected from previousstudies, the GC147 target is heavily phosphorylated, and theKu polypeptides (p70 >> p80) and p350 are autophosphory-lated (27). The necessity ofindividual factors for formation ofan active DNA-PK complex is shown in Fig. SB. Assemblycomponents were combined in the presence of [y-32P]ATP, orDNA and Ku protein were separately omitted. Antibodies toKu, p350, and RNAP II immunoprecipitated the GC147ophosphorylation target when all components of the complexwere combined. In the absence of either Ku protein or DNA,catalytic activity was either minimal or absent. These resultsdemonstrate that catalytically active complexes ofDNA-PKare sufficiently stable to undergo immunoprecipitation, andthey suggest that the complexes detected in HeLa cellextracts are also catalytically active.

DISCUSSIONThe present observations extend recent insights into thebiological function of the Ku protein through the demonstra-tion that this protein assembles into a catalytically activecomplex with p350 in the presence of linear double-strandedDNA. This is the DNA form that activates DNA-PK andinteracts with Ku protein, initially at its free ends (9, 25, 26).These results are consistent with earlier observations of Dviret al. (16) showing that p350 induces a supershift in Kuprotein-DNA gel mobility and those of Gottlieb and Jackson(15) demonstrating the presence of Ku protein and p350 insome immunoprecipitates prepared with anti-Ku antibodies.The DNA-PK complex is highly sensitive to the ionic

strength of the environment, as are many other structuresthat involve DNA-protein interactions. This complex wasstable at 0.15 M NaCI but tended to dissociate progressivelyas the salt concentration was increased from 0.2 to 0.3 MNaCl. In the previous studies, we noted that Ku proteindissociates completely from DNA in buffers containing 0.35M NaCl (9). Thus, the interaction between p350 and Kuprotein-DNA binding site may be somewhat less stable thanthe interaction of Ku protein with DNA. Most likely, this

46 -

B DNAAUUASKu

CD~~~~~~~0

=6 CZ CZ 1C CZ

kDa

200 -

97.4 -

69 -

- p350

t - GC147o

- GC147a

FIG. 5. Immunoprecipitable Ku protein-p350 complexes arecatalytically active. (A) Biochemically purified Ku protein, p350, thetarget protein GC147, and the DNA template AdUAS were incubatedtogether in the presence of ('y-32P]ATP and immunoprecipitated withvarious antibodies as shown. GC147o is the hyperphosphorylatedform ofGC147a. The latter is visualized because it is phosphorylitedat only a few sites. (B) All constituents were combined (lanes 1-3) orKu protein (lanes 4-6) and DNA (lanes 7-9) were omitted as shown.Immunoprecipitations were performed with the indicated antibodies.The anti-p350 antiserum was a polyclonal mouse serum prepared inour laboratory. This serum is monospecific for p350 in immunopre-cipitations carried out at 0.5 M NaCl and coprecipitates Ku proteinin studies performed at 0.15 M NaCl.

complex was not observed in earlier immunoprecipitationexperiments because of its instability in the buffers ordinarilyused for these studies (5, 28). The high level of solubility ofthe Ku protein-p350-DNA structures is likely to reflect theirrelease from intact chromatin as DNA is sheared in thecourse of cell sonification. Indeed, such cell extracts may beenriched in transcriptionally active chromatin fiagmentssince the relatively open chromatin structure within suchregions may enhance susceptibility to fragmentation andsolubilization.

Several arguments indicate that Ku protein and p350 areclosely approximated on the DNA strand. The previousstudies demonstrated that UV light crosslinks p350 to.DNAonly in the presence of Ku protein (15). The present studiesdemonstrated that the Ku protein-p350 complex resists ex-tended DNase I digestion and exposure to EtdBr, an agentthat disrupts mainly DNA-protein interaction. These latterstudies imply either that Ku protein and p350 are physicallyassociated even after dissociation from the DNA strand orthat very small DNA figments serve to stabilize this com-plex. We would add the caveat that we cannot exclude the

A i L;a c:

kDa

200-

97.4 -

69 -

-- P350

*- GC14To*- GO147a-p80-- p70

6907Biochemistry: Suwa et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

possibility that both Ku protein and p350 have unusual abilityto bind to DNA that has intercalated EtdBr. These observa-tion support a model in which Ku protein associates with anappropriate DNA domain to create a binding site for a p350molecule.An important question now is, how does the Ku protein

recognize a DNA site that is poised for transcription? Inearlier studies, we noted that Ku protein binds well to lineardouble-stranded DNA (9), which is the DNA form requiredfor DNA-PK activity in vitro (25, 26). However, it does notseem likely that free DNA ends regularly occur at transcrip-tion sites. Some evidence suggests that the Ku protein canslide along the DNA strand, and it is possible that it associ-ates with DNA at some distal site and traverses along theDNA until an initiation complex is encountered (10). Thatconcept would pose the problem of how Ku protein mighttraverse a DNA region bearing at least some nucleosomalconfigurations. It should also be noted that the Ku proteinrecognizes DNA forms other than those with free ends. Forexample, we have recently found that Ku protein binds welltoDNA nick sites (12). However, nicked circular DNA is nota good activator of DNA-PK activity (25, 26). It is possiblethat Ku protein bound to such sites might interact withproteins other than p350 such as DNA repair components.Binding of Ku protein to DNA hairpin loops has also beennoted (13), but again it is not clear that such DNA structuresrelate to activation of transcription. Most recently, thisprotein was noted to bind to DNA minicircles bearing mis-matched (bubble) segments of 30 bp and to prefer DNA endsbearing A-T rather than G-C termini, suggesting that itrecognizes single- to double-stranded DNA transition sites inpromoter regions or transcription bubbles (35). Thus, itappears that Ku protein utilizes its ability to recognizespecific DNA forms to localize p350 to sites of active geneexpression.

We thank Drs. William S. Dynan and Arik Dvir for helpful adviceand discussions, Dr. Westley H. Reeves for monoclonal antibodiesto the Ku protein, and Dr. Stephen P. Jackson for rabbit polyclonalantibodies to p350. This work was supported by National Institutesof Health Grants AR32549 (to J.A.H.) and GM35866 (to W. S.Dynan, University of Colorado), a Biomedical Research Grant fromthe Arthritis Foundation, funds from the Georgia Research Alliance,and a generous donation from the Matuzak family.

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6908 Biochemistry: Suwa et al.