Identification of Two Domains of the P70 Ku Protein Mediating Dimerization with P80 and DNA Binding*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 2, Issue of January 9, pp. 842–848, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Identification of Two Domains of the p70 Ku Protein Mediating Dimerization with p80 and DNA Binding*

(Received for publication, August 27, 1997)

Jingsong Wang‡, Xingwen Dong‡, Kyungjae Myung§, Eric A. Hendrickson§¶, and Westley H. Reevesʈ From the Departments of Medicine, Microbiology and Immunology, Thurston Arthritis Research Center, UNC Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7280 and the §Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912

The Ku autoantigen is a heterodimer of 70 (p70) and strand transitions (reviewed in Ref. 1). Sequence-specific DNA ϳ80 kDa (p80) subunits that is the DNA-binding compo- binding also has been reported (1, 4). Using DNA immunopre- nent of the DNA-dependent protein kinase (DNA-PK) cipitation and Southwestern blot assays, the p70 protein has complex involved in DNA repair and V(D)J recombina- been shown to interact with DNA without requiring p80 (5–7), tion. Binding to DNA ends is critical to the function of and a DNA binding domain was mapped to the C-terminal DNA-PK, but how Ku interacts with DNA is not com- region (p70, amino acids 536–609) (7, 8). However, in gel shift pletely understood. To define the role of p70 and p80 and assays, dimerization of p70 with p80 is required for DNA bind- their dimerization in DNA binding, heterodimers were ing (9, 10). Determining how p70 and p80 interact with each assembled by co-expressing the subunits using recombi- other and with DNA termini or with specific sequences is nant baculoviruses. Two p70 dimerization sites, amino critical to understanding the functions of Ku in DNA repair, acids 1–115 and 430–482, respectively, were identified. V(D)J recombination, and transcriptional activation. To better Binding of p70 to linear double-stranded DNA could be understand the role of p70/p80 dimerization in DNA binding, demonstrated by an immunoprecipitation assay, and re- p70 deletion mutants expressed in Sf9 cells using recombinant quired the C-terminal portion (amino acids 430–609), but not interaction with p80. The p70 mutants 1–600, baculoviruses were used to identify functional sites of the p70 1–542, 1–115, and 430–600 did not bind DNA efficiently. subunit. However, DNA binding of 1–600, 1–542, and 1–115, but EXPERIMENTAL PROCEDURES not 430–600, was restored by dimerization with p80, Cells and Viruses—K562 (human erythroleukemia, from the Ameri- indicating that p70 has two DNA binding sites, each can Type Culture Collection, ATCC, Rockville, MD) were maintained in partially overlapping one of the dimerization sites. The RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/ C-terminal domain can bind DNA by itself, but the N- streptomycin. The Sf9 (Spodoptera frugiperda ovary) cell line was ob- terminal domain requires dimerization with p80. These tained from the ATCC and maintained at 27 °C in Grace’s insect tissue observations could be relevant to the multiple func- culture medium supplemented with 3.3 g/liter TC yeastolate, 3.3 g/liter tional activities of Ku and explain controversies regard- lactalbumin hydrolyzate (University of North Carolina Lineberger ing the role of dimerization in DNA binding. Comprehensive Cancer Center Tissue Culture Facility), 10% fetal bovine serum, and penicillin/streptomycin. Recombinant Baculoviruses—The construction of transfer vectors and recombinant baculoviruses expressing full-length human Ku p70 The Ku (p70/p80) antigen was first identified and character- and p80 has been described (11). Briefly, p70 and p80 Ku cDNAs were ized as a DNA binding autoantigen recognized by antibodies subcloned into a modified baculovirus transfer vector pVLB4-mp53 (11) present in the sera of certain patients with scleroderma-poly- based on pVL1392 (PharMingen, San Diego, CA) and pBlueBacHisA (Invitrogen Corp., San Diego, CA). The recombinant vectors express Ku myositis overlap syndrome or systemic lupus erythematosus proteins fused to an N-terminal polyhistidine sequence and enteroki- (SLE) (1). It was later shown to be the DNA binding component nase cleavage site. Transfer vectors were co-transfected with linearized of a DNA-dependent protein kinase that phosphorylates sev- wild-type baculovirus (BaculoGoldTM, PharMingen), into Sf9 cells. Re- eral chromatin-bound proteins in vitro and is involved in V(D)J combinant baculovirus clones were selected by end point dilution assay. recombination and double-stranded (ds)1 DNA break repair (2, Recombinant baculoviruses expressing the full-length p80 or p70 are 3). Ku antigen, a heterodimer of 70- (p70) and ϳ80-kDa (p80) designated p80-bv and p70-bv, respectively. Recombinant baculoviruses expressing mutant p70 proteins were subunits, binds to dsDNA termini, nicks, or single to double constructed in the same manner. A cDNA sequence encoding p70 amino acids 1–600 (7) was subcloned into the BamHI site of pVL1393 and inserted by homologous recombination into baculovirus. The recombi- * This work was supported in part by Grants R01-AR40391 (to nant baculovirus permits expression of an untagged p70 (amino acids W. H. R.), R01-AI35763 (to E. A. H.), and T32-AR7416 from the United 1–600) protein. The cDNAs for additional p70 mutants were amplified States Public Health Service. The costs of publication of this article by polymerase chain reaction using primer pairs listed in Table I, and were defrayed in part by the payment of page charges. This article must subcloned into pVLB4-mp53 at the BamHI-SmaIorBamHI-EcoRI sites. therefore be hereby marked “advertisement” in accordance with 18 Expression of Recombinant Proteins—Sf9 cells were infected with U.S.C. Section 1734 solely to indicate this fact. recombinant baculoviruses at m.o.i. of 10 as described (11). For co- ‡ Supported by a Postdoctoral Fellowship from the Arthritis infection, Sf9 cells were infected with two recombinant baculoviruses Foundation. ¶ Leukemia Scholar of America. each at m.o.i. of 10. 72 h after infection, recombinant p70 or p80 Ku ʈ To whom correspondence should be addressed: Division of Rheuma- proteins were identified in Sf9 cell lysates by SDS-PAGE and immuno- tology and Immunology, University of North Carolina at Chapel Hill, blotting using human autoimmune serum and specific monoclonal 3330 Thurston Bldg., CB 7280, Chapel Hill, NC 27599-7280. Tel.: 919- antibodies. 966-4191; Fax: 919-966-1739; E-mail: [email protected]. Monoclonal Antibodies—Murine monoclonal antibodies (mAbs) spe- 1 The abbreviations used are: ds, double-stranded; mAb, monoclonal cific for the human Ku antigen were characterized previously (1). Their antibody; PAGE, polyacrylamide gel electrophoresis; m.o.i., multiplicity isotypes and specificities are as follows: 162, IgG2a specific for the of infection; NET, NaCl-EDTA-Tris; bv, baculovirus. p70/p80 dimer (unreactive with free p70 or p80); 111, IgG1 specific for

842 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Functional Domains of Ku Antigen 843

TABLE I Primers used to amplify Ku cDNAs for subcloning into baculovirus transfer vectors

Constructs 5Ј Primer 3Ј Primer p80.1–732 (full length) 5Ј-GCGGATCCATGGTGCGGTCGGGGAATAAG-3Ј 5Ј-GCGAATTCCGACCTATATCATGTCC-3Ј p70.1–609 (full length) 5Ј-GCGGATCCATGTCAGGGTGGGAGTC-3Ј 5Ј-GCCCGGGTCAGTCCTGGAAGTGCTTG-3Ј p70.505–609 5Ј-GCGGATCCGACCTGACATTGCCCAAG-3Ј 5Ј-GCCCGGGTCAGTCCTGGAAGTGCTTG-3Ј p70.480–609 5Ј-GCGGATCCAACCCCGTGCTGCAGCAG-3Ј 5Ј-GCCCGGGTCAGTCCTGGAAGTGCTTG-3Ј p70.430–609 5Ј-GCGGATCCCCAGGCTTCCAGCTGGTC-3Ј 5Ј-GCCCGGGTCAGTCCTGGAAGTGCTTG-3Ј p70.380–609 5Ј-GCGGATCCACCCTGTTCAGTGCTCTG-3Ј 5Ј-GCCCGGGTCAGTCCTGGAAGTGCTTG-3Ј p70.330–609 5Ј-GCGGATCCGAGAAAGAGGAAACAGAAG-3Ј 5Ј-GCCCGGGTCAGTCCTGGAAGTGCTTG-3Ј p70.430–600 5Ј-GCGGATCCCCAGGCTTCCAGCTGGTC-3Ј 5Ј-GCCCCGGGTCAATCCAGCAGCTCCTGCTTC-3Ј p70.1–542 5Ј-GCGGATCCATGTCAGGGTGGGAGTC-3Ј 5Ј-GCCCCGGGTCACTTGGTAACTTTCCCTTC-3Ј p70.430–542 5Ј-GCGGATCCCCAGGCTTCCAGCTGGTC-3Ј 5Ј-GCCCCGGGTCACTTGGTAACTTTCCCTTC-3Ј p70.461–542 5Ј-GCGGATCCAAGATGAAGGCTATCGTTG-3Ј 5Ј-GCCCCGGGTCACTTGGTAACTTTCCCTTC-3Ј p70.430–482 5Ј-GCGGATCCCCAGGCTTCCAGCTGGTC-3Ј 5Ј-GCCCCGGGTCACACGGGGTTCTCAAAGC-3Ј p70.1–433 5Ј-GCGGATCCATGTCAGGGTGGGAGTC-3Ј 5Ј-GCCCCGGGTCACTGGAAGCCTGGAGGAGTC-3Ј p70.244–433 5Ј-GCGGATCCCGGAAGGTTCGCGCCAAG-3Ј 5Ј-GCCCCGGGTCACTGGAAGCCTGGAGGAGTC-3Ј p70.116–266 5Ј-GCGGATCCATTCTAGAGCTTGACCAG-3Ј 5Ј-GCCCCGGGTCAATCTTTGTTGAGCTTCAG-3Ј p70.1–214 5Ј-GCGGATCCATGTCAGGGTGGGAGTC-3Ј 5Ј-GCCCCGGGTCAGGATATGTCAAAGCCCC-3Ј p70.1–115 5Ј-GCGGATCCATGTCAGGGTGGGAGTC-3Ј 5Ј-GCGAATTCTCATCGTTTTGCACCTGG-3Ј

human p80 (amino acids 610–705); N3H10, IgG2b specific for p70 ured as described (8) with minor modifications. Cell extract from Sf9 (amino acids 506–541). Murine mAbs specific for the polyhistidine tag cells infected with recombinant baculoviruses was immunoprecipitated HIS (IgG2a) and anti-Xpress (IgG1) were obtained from Sigma Chem- with 3 ␮l of mAbs 162, N3H10, 111, or anti-Xpress ascites. Approxi- ical Co. (St. Louis, MO) and Invitrogen Corp., respectively. mately equal amounts of the different Ku proteins were bound to the Immunoblotting—Immunoblot analysis of the recombinant p70 and beads, as determined by SDS-PAGE with Coomassie Blue staining in p80 proteins from Sf9 cells or Ku proteins from K562 cells was per- preliminary experiments. Beads were washed with 1.5 M NaCl NET- formed using human autoantibodies or murine mAbs as described (11). Nonidet P-40 buffer followed by the same buffer containing 50 mM NaCl Ascitic fluid from mAbs N3H10, S10B1, and 111 or anti-Xpress was instead of 1.5 M NaCl. Radiolabeled DNA probe (25 ng) was added for diluted 1:1000 for probing immunoblots, followed by 1:1500 alkaline 1 h, and the beads were washed again with NET-Nonidet P-40 buffer. phosphatase-conjugated goat anti-mouse IgG antibodies (Tago, DNA bound to the purified Ku antigen was recovered by digesting the Burlingame, CA). beads with proteinase K, and an aliquot of the supernatant was used for Immunoprecipitation—Radiolabeling of baculovirus-infected Sf9 scintillation counting. cells with [35S]methionine/cysteine and immunoprecipitation was car- Gel Mobility Shift Assay—A gel mobility shift assay for determining ried out as described (11). Briefly, radiolabeled cells were lysed in Ku DNA end binding activity (13) was used with minor modifications. NET-Nonidet P-40 buffer (0.15 M NaCl, 50 mM Tris, pH 7.5, 0.3% Radiolabeled DNA was incubated with whole-cell extracts in 20 ␮lof Nonidet P-40, 2 mM EDTA) with 0.5 mM phenylmethylsulfonyl fluoride binding buffer (10 mM Tris, pH 7.5, 1 mM DTT, 5 mM EDTA, 10% and aprotinin (0.3 trypsin inhibitor units/ml; Sigma). Cleared lysate glycerol, 150 mM NaCl, 2 ␮g of closed circular plasmid ␾X174 Rf I DNA was incubated for 2 h with mAb-coated protein A-Sepharose beads at (New England Biolabs as a nonspecific inhibitor) at 22 °C for 30 min. 4 °C. The beads were then washed three times with 1.5 M NaCl NET- The samples were subjected to electrophoresis in a 4% polyacrylamide Nonidet P-40 buffer (1.5 M NaCl, 50 mM Tris, pH 7.5, 0.3% Nonidet P-40, gelat22°Cfor4hat100V.Thegelwasdried and autoradiographed 2mM EDTA) and once with NET buffer (0.15 M NaCl, 50 mM Tris, pH at Ϫ70 °C. For supershift experiments, mAbs (1 ␮l of ammonium sul- 7.5, 2 mM EDTA). Immunoprecipitated proteins were eluted from the fate precipitated ascitic fluid at ϳ1 mg/ml) were added to the binding beads by boiling in SDS sample buffer, and analyzed by SDS-PAGE. mixture 5 min after adding probe and incubated for an additional 30 Limited Protease Digestion—The p70/p80 Ku heterodimer from K562 min at 22 °C before electrophoresis. cells was affinity purified onto mAb 162-coated protein A-Sepharose beads as described (7, 8). Protease V8 (Boehringer Mannheim), trypsin- RESULTS TPCK (Millipore Corporation, Freehold, NJ), or chymotrypsin (Worth- ington Biochemical Corporation, Freehold, NJ) was then added to the The human p70 and p80 Ku proteins assemble into het- ␮ beads at 2 to 200 g/ml in 10 mM Tris-HCl, pH 7.5, and 2.5 mM CaCl2 erodimers in human cells as well as insect cells infected with for 30 min at 22 °C. After incubation, the beads were washed three recombinant baculoviruses expressing the human Ku subunits times with NET-Nonidet P-40 buffer and once with NET buffer and (11, 14). The C-terminal portion of p70 protein contains a then subjected to SDS-PAGE and Western blotting. The nitrocellulose leucine zipper-like sequence (15), but its role in interactions membrane was probed with N3H10 to detect the p70 remaining associated with p80 in the 162 immunoprecipitates. The sizes of p70 frag- with p80 is not established. In the present study, dimerization ments were determined by comparing with molecular weight standards. of full-length p80 with p70 deletion mutants in extracts from Cell Extracts—Whole-cell extracts were prepared from Sf9 or K562 co-infected Sf9 cells was investigated using the following crite- cells as described (12). Cells were washed with hypotonic buffer (10 mM ria: 1) co-immunoprecipitation of p70 by anti-p80 mAbs and HEPES, pH 7.3, 25 mM KCl, 10 mM NaCl, 1 mM MgCl , 0.1 mM EDTA), 2 vice versa, and 2) immunoprecipitation of both p70 and p80 by and pellets were resuspended at 2 ϫ 107 cells/100 ␮l in the same buffer mAb 162, which recognizes the p70/p80 heterodimer but not supplemented with 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, and 0.5 ␮g/ml aprotinin. Cells were lysed by three the individual subunits.

freeze-thaw cycles and then adjusted to 0.5 M KCl and 10 mM MgCl2. C-terminal Dimerization Domain—To help define the role of After centrifuging (10,000 ϫ g for 10 min at 4 °C), the supernatant was the leucine zipper-like sequence of p70 identified previously recovered, diluted to 0.1 M KCl by adding 50 mM HEPES, pH 7.3, and (15) in dimerization, deletion mutants were constructed (Fig. Ϫ stored at 80 °C. 1A) and co-expressed with full-length p80 in Sf9 cells using the Radiolabeled Linear DNA Probe—Preparation of the DNA probe used for the DNA immunoprecipitation assay and electrophoretic mo- baculovirus system. Expression was verified by immunopre- bility shift assay was described previously (8). Briefly, a 564-base pair cipitation using a polyhistidine-specific mAb (Fig. 1B). To de- linear double-stranded DNA fragment generated by HindIII digestion tect heterodimer formation, the p70 mutants were co-immuno- of bacteriophage ␭ DNA (New England Biolabs, Beverly, MA) was precipitated with anti-p80 mAb 111 (Fig. 1B). In agreement 32 end-labeled with [ P]dATP (3,000 Ci/mmol, NEN Life Science Prod- with previous observations (11), full-length p70 was co-immu- ucts) using Klenow fragment (Boehringer Mannheim). For gel shift noprecipitated by mAb 111. Mutant p70 proteins 330–609, assays, the radiolabeled probe was purified using a Geneclean III kit from Bio 101 (Vista, CA). 380–609, and 430–609 also were co-immunoprecipitated by DNA Immunoprecipitation Assay—The binding of radiolabeled DNA mAb 111. In contrast, the mutant proteins 480–609 and 505– to affinity purified Ku antigen on protein A-Sepharose beads was meas- 609 were not co-immunoprecipitated. These data suggest that a 844 Functional Domains of Ku Antigen

FIG.1.Mapping of a dimerization domain of p70. A, diagram of the p70 deletion mutants tested in panel B. Amino acid positions of the p70 are indicated on the right. Expressed portions of p70 are shaded black. Location of the p70 leucine zipper-like sequence is indicated by cross-hatching. Ability to be co-immunoprecipitated by mAb 111 (Dimerization) also is indicated. B, co-immunoprecipitation of p70 deletion mutants with p80. Sf9 cells were co-infected with p80-bv plus FIG.2.Minimal C-terminal dimerization domain of p70. A, di- p70-bv (1–609) or p70 deletion mutants (amino acids 330–609, 380– agram of the p70 deletion mutants tested in panel B. Amino acid 609, 430–609, 480–609, 505–609), as indicated. Cells were radiola- positions of the p70 are indicated on the right. Expressed portions of beled, and extracts were immunoprecipitated with a polyhistidine-spe- p70 are shaded black. Location of the p70 leucine zipper-like sequence cific mAb (Xpress) that recognizes all of the recombinant histidine- is indicated by cross-hatching. Ability to be co-immunoprecipitated by tagged proteins or with mAb 111 (anti-p80) followed by SDS-PAGE and mAb 111 (Dimerization) also is indicated. B, co-immunoprecipitation of autoradiography. Positions of p80, full-length p70 (1–609 lane), and the p70 deletion mutants with p80. Immunoprecipitations with anti-poly- p70 deletion mutants are indicated by arrows. histidine (Xpress) and anti-p80 (111) mAbs were carried out as in the legend to Fig. 1. Co-immunoprecipitation of p70 deletion mutants consisting of amino acids 1–600, 430–600, 1–542, 430–542, 461–542, and dimerization domain is located on the C-terminal portion of p70 430–482 along with p80 was examined. Positions of full-length recom- (amino acids 430–609) and that the leucine zipper-like region binant (histidine-tagged) p80 and the p70 deletion mutants are indi- (amino acids 483–504) is not sufficient for dimerization with cated by arrows. p80. To further localize this domain, a second panel of deletion but not 1–433, localizing the mAb 162 epitope to the C-terminal mutants (Fig. 2A) was co-expressed with p80. As before, protein portion of p70. Further deletions revealed that mAb 162 could expression was verified by immunoprecipitating with anti- immunoprecipitate p80 when it was co-expressed with 430–542 Xpress (Fig. 2B, Xpress), and dimer formation was demon- but not with 461–542. mAb 162 did not immunoprecipitate p80 or strated by co-immunoprecipitating with mAb 111 (Fig. 2B, any of the p70 deletion mutants when expressed alone in Sf9 cells 111). Although deletion of amino acids 601–609 abrogates the (Ref.16 and data not shown). These data indicate that mAb 162 DNA binding activity of p70 in immunoprecipitation and recognizes an epitope formed by p80 plus amino acids 430–542 of Southwestern blot assays (7), deleting this sequence had no p70. This region overlaps the C-terminal dimerization domain apparent effect on dimerization (Figs. 3, A and B, 1–600 and identified in Figs. 1 and 2 as well as a strongly basic sequence 430–600). The mutants 1–542 and 430–542 also dimerized adjacent to a leucine zipper-like region. efficiently with p80. Unexpectedly, so did the 430–482 frag- Localization of the Dimerization Site by Limited Proteoly- ment (Figs. 2, A and B). These data indicate that the leucine sis—To further verify the results obtained using recombinant zipper-like sequence of p70 was not required for dimerization baculoviruses directing the synthesis of p70 and p80, dimeriza- with p80 and that the minimal dimerization site is located on tion was evaluated in human K562 cells. Ku heterodimer was amino acids 430–482. affinity purified onto beads with mAb 162, followed by protease Antigenic Site Recognized by mAb 162—The p70/p80 het- treatment of the beads. mAb 162 recognizes an epitope com- erodimer is recognized specifically by mAb 162 and protected prised of p70 plus p80 and stabilizes the dimer under dissoci- from dissociation in the presence of 1.5 M NaCl and detergent ating conditions. Since the binding of antibodies frequently (16), suggesting that mAb 162 may bind to an antigenic deter- protects proteins from proteolytic cleavage (17, 18), we ex- minant near the p70/p80 dimerization site. The relationship of pected that the dimerization site-associated epitope recognized the epitope recognized by 162 to the dimerization site identified by 162 might be protected from degradation. in Figs. 1–2 was investigated by immunoprecipitating p70 de- The p70 fragments retained on mAb 162 beads after protease letion mutants co-expressed with full-length p80 (Fig. 3A). The treatment were analyzed by Western blot using mAb N3H10 level of recombinant protein expression was verified by anti- (Fig. 4). The calculated sizes of the smallest p70 fragments Xpress antibody (Fig. 3B). mAb 162 immunoprecipitated full- recognized by N3H10 after trypsin or chymotrypsin digestion length p80 when it was co-expressed with p70 mutant 430–609, were 14 and 16 kDa, respectively (Fig. 4, lower arrow in each Functional Domains of Ku Antigen 845

FIG.4.Limited protease digestion of 162 affinity purified Ku antigen from K562 cells. Extract from unlabeled K562 cells was affinity purified onto mAb 162 (anti-p70/p80 dimer) coated protein A-Sepharose beads. Protease V8, trypsin, or chymotrypsin were then added to the beads at 2 (lane 1), 20 (lane 2), or 200 (lane 3) ␮g/ml. After digestion, the beads were washed 3 times with NET-Nonidet P-40 buffer and once with NET buffer and subjected to SDS-PAGE and Western blotting. The nitrocellulose membrane was probed with N3H10 (anti-p70, amino acids 506–541). The fragments of p70 associated with the beads after washing that were recognized by N3H10 are indicated by arrowheads. Positions of molecular mass standards (kDa) are indicated on the right.

FIG.3.p70 contribution to mAb 162 recognition of Ku. A, diagram of the recombinant p70 deletion mutants tested in panel B. Amino acid positions of p70 are indicated on the right. Expressed portions of p70 are shaded black. Location of the p70 leucine zipper-like sequence is indicated by cross-hatching. B, immunoprecipitation. Sf9 cells were co-infected with recombinant baculoviruses expressing full-length p80 plus p70 deletion mutants 1–433, 430–609, 430–542, and 461–542, respectively. The cells were radiolabeled with [35S]methionine, and extracts were immunoprecipitated with an anti-polyhistidine mAb (HIS) that recognizes the N-terminal histidine tag of the recombinant p80 protein, as well as the p70 deletion mutants, or with mAb 162 (specific for the dimer of p70 with p80). Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Position of full-length p80 is indicated by an arrowhead. lane). Since the N3H10 epitope has been localized to amino acids 529–541 of p70,2 these studies lend further support to the idea that a dimerization site is located near the C terminus of p70. Identification of Second Dimerization Domain Near the N Terminus of p70—The identification of a p80 dimerization domain near the C terminus of p70 did not exclude the possibility that additional sequences also could mediate interactions between p70 and p80. To examine that possibility, p70 mutant 1–433 (Fig. 5A) was co-expressed with p80, followed by immunoprecipitation with mAb 111. This fragment dimerized with p80 (Fig. 5B). Mutants 1–214 and 1–115 also were co-immunoprecipitated by mAb 111, but mutants 244–433 and 116–266 FIG.5.Mapping of an N-terminal dimerization domain of p70. were not, suggesting that the second dimerization domain is A, diagram of the p70 deletion mutants tested in panel B. Amino acid located on the N-terminal portion of p70 (Fig. 5A). Thus, two positions of the p70 are indicated on the right. Expressed portions of p70 are shaded black. Ability to be co-immunoprecipitated by mAb 111 discrete domains mediate interactions of p70 with p80. Of note, (Dimerization) also is indicated. B, co-immunoprecipitation of p70 de- mAb 162 did not recognize the dimer of p80 with p70 (1–433) letion mutants with p80. Immunoprecipitations with anti-polyhistidine (Fig. 3B), suggesting that it is specific for the C-terminal dimer- (Xpress) and anti-p80 (111) mAbs were carried out as in the legend to ization site. Fig. 1. Co-immunoprecipitation of p70 deletion mutants consisting of amino acids 1–433, 244–433, 116–266, 1–214, and 1–115 along with Effects of Dimerization on the Binding of Ku to DNA in p80 was examined. Position of full-length recombinant (histidine- Immunoprecipitation Assay—Since it has been reported that tagged) p80 and the p70 deletion mutants are indicated by arrows. p70/p80 dimerization is critical for the DNA binding activity of Ku in gel shift assays (9, 10), but not DNA immunoprecipitation or Southwestern blot assays (5–7), we examined the role of each of the two dimerization sites in the interaction of Ku with 2 J. Wang, unpublished data. DNA. The C-terminal portion of p70 binds readily to linear 846 Functional Domains of Ku Antigen

FIG.6. DNA immunoprecipitation assay. Cell extract from Sf9 cells infected with wild-type baculovirus (WT) or recombinant baculoviruses expressing full-length (1–609) or mutant p70 as indicated were immunoprecipitated with mAbs N3H10 (A), or HIS (B). Approxi- mately equal amounts of affinity purified Ku antigens, as determined in preliminary experiments, were used in each assay. Binding of the affinity purified proteins to a 32P-radiolabeled linear bacteriophage ␭ HindIII DNA fragment was evaluated. Binding of each p70 construct to the radiolabeled DNA probe was tested alone (hatched bars) as well as after dimerization to full-length p80 (from extracts of cells co-infected with a p70 construct plus full-length p80-bv, indicated by solid bars). dsDNA fragments in a DNA immunoprecipitation assay (7). In agreement with those previous observations, recombinant full- length p70 (1–609) bound linear dsDNA comparably with recombinant p70/p80 dimer (Fig. 6B). As shown previously (7), deleting the C-terminal 9 amino acids (mutant 1–600) reduced the DNA binding activity (Fig. 6A). The importance of an intact C terminus was underscored by the fact that p70 mutants 380–609 and 430–609 had DNA binding activity similar to that of wild-type p70/p80 heterodimer (Fig. 6B). Consistent with this observation, p70 mutants lacking larger portions of the C terminus had low DNA binding activity (Fig. 6B, mutants 1–542, 1–115, and 430–600). Mutants 430–542 and 430–482 also had little binding activity (not shown). Un- expectedly, when p80 was co-expressed with mutant construct 1–542, DNA binding activity was restored to nearly normal, whereas partial restoration was seen with 1–600 and 1–115 (Figs. 6A and B). However, dimerization with p80 had no effect on the DNA end binding activity of p70 mutant 430–600 (Fig. 6B). Consistent with previous observations (5–7), p80 alone did not bind DNA (not shown), indicating that there was little or no dimerization of the recombinant human p80 with endogenous insect p70. Together, these results strongly suggest that, like dimerization, the binding of Ku to DNA in immunoprecipitation assays is mediated by two separate sites. One site, located at the C terminus of p70, does not depend on interactions with p80. The second, N-terminal, site requires dimerization of p80 with the N-terminal dimerization domain of p70. Effect of Dimerization on DNA Binding in Gel Mobility Shift Assay—Different requirements for p80 have been found in gel mobility shift assays (dimerization-dependent) than in immu- FIG.7.DNA binding of Ku proteins by gel mobility shift assay. noprecipitation or Southwestern blot assays (dimerization-in- A, binding of mutant Ku antigens to DNA. The same radiolabeled linear dependent). To investigate the basis for this discrepancy, bind- DNA probe used for the DNA immunoprecipitation assay (Fig. 6) was ing of recombinant human Ku to DNA was investigated in a gel incubated with extracts from Sf9 cells infected with full-length or mu- mobility shift assay (Fig. 7) using the same probe as in the tant p70 (430–609, 1–600, or 1–542), with or without full-length p80, as indicated. Extract from human K562 cells also was tested as well as free immunoprecipitation assay (Fig. 6). probe without any cell extract (lane P). Binding mixtures were resolved Consistent with previous findings (9, 10), recombinant Ku by electrophoresis on a 4% polyacrylamide gel, followed by autoradiog- heterodimer expressed in Sf9 cells using recombinant baculo- raphy. The position of free probe (FP) as well as complexes formed with viruses bound DNA efficiently, whereas full-length free p70 extracts from control K562 cells and infected Sf9 cells (B1-B4) are indicated. B, supershift of DNA-protein complexes with mAbs. Gel and p80 both failed to bind DNA in the gel shift assay (Fig. 7A). mobility shift assays were performed as in panel A, but mAb 162 Interestingly, p70 mutant 1–600 with an intact N-terminal (anti-p70/p80 dimer) or anti-HIS mAb was included in some reaction and partially deleted C-terminal DNA binding domain did not mixtures, as indicated. Bands supershifted by addition of mAbs are bind DNA alone, whereas upon dimerization with p80, DNA shown. binding activity was restored (Fig. 7A). Furthermore, the multi-band pattern visible on the gel (Fig. 7A, 1–600/p80 lane) the two DNA binding domains, did not bind DNA in gel shift suggests that dimerization also restores the internal translo- assays, even after dimerization with p80 (Fig. 7A). Binding of cation property of Ku (19). A similar, but possibly smaller, the recombinant mutant Ku antigens p70/p80 and 1–600/p80 effect was seen in the DNA immunoprecipitation assay (Fig. to DNA was verified by supershift assays using anti-Ku mAb 6A). The p70 mutants 1–542 and 430–609, each carrying one of 162 and an antibody to the polyhistidine tag of the recombinant Functional Domains of Ku Antigen 847

mune disease patients’ sera that, like mAb 162, also recognize and stabilize the p70/p80 heterodimer (11). Interestingly, some of these sera appeared to recognize a different epitope than that bound by mAb 162 since they do not compete with mAb 162 for binding to Ku. This raises the possibility that additional subunit interaction domains may be present. The identification of a second dimerization site on amino acids 1–115 of p70 (Fig. 5) is consistent with that possibility. The corresponding dimerization sites of p80 have yet to be defined precisely although the C-terminal 32 kDa of p80 has been reported to exhibit p70 binding activity (10). More precise localization of the p70 interaction sites on p80 is in progress. The role of the leucine zipper-like motif of p70, if any, re- FIG.8.DNA binding and p80 interaction domains of p70. At the top is a diagram of the full-length p70 protein showing the location of a mains to be determined. The present studies show conclusively leucine zipper-like sequence (amino acids 483–504) previously identi- that this region is not required for p70-p80 interactions. Other fied by sequence analysis. Locations of two sites responsible for dimer- data suggest that it is not required for interactions of p70 with ization with p80 (amino acids 1–115 and 430–482, respectively) are p90vav (20). The catalytic subunit of DNA-dependent protein indicated beneath in solid shading. Note that the leucine zipper-like sequence is not required. Locations of two regions involved in DNA kinase contains an extensive leucine zipper-like sequence (21) binding are shown (cross-hatched). The C-terminal region (amino acids that possibly might interact with the leucine zipper-like motif 536–609), which binds DNA independently of p80 interactions, was of p70. Alternatively, this sequence might interact with other localized previously (7). An N-terminal region, requiring amino acids proteins or not be of functional significance. 1–542 for full activity and 1–115 for partial activity, mediates DNA binding only after dimerizing with p80. Finally, a region that forms the Mechanism of DNA Binding—Controversy remains about epitope recognized by mAb 162 after dimerizing with p80 is shown at the role of p80 in DNA binding. Gel shift assays suggest that the bottom. dimerization of p70 with p80 is required for DNA binding (9, 10). In contrast, DNA immunoprecipitation and Southwestern proteins (Fig. 7B). Taken together, the data suggest that Ku blot assays suggest that p70 by itself interacts with DNA ter- DNA binding activity in the gel shift assay requires that both mini, whereas p80 does not (5–7). The present data may ex- the N- and C-terminal DNA binding domains of p70 be intact. plain this discrepancy. DNA immunoprecipitation assays indi- Since p80 is needed for activity of the N-terminal DNA binding cate that two different regions of p70 are involved in DNA site, it is required for binding in the gel shift assay. Finally, the binding. One is the previously identified C-terminal DNA bind- data suggest that dimerization can compensate partially for ing site (7). This site is carried by p70 mutant 430–609, and its deletion of a portion (amino acids 601–609) of the C-terminal DNA binding activity is unaffected by dimerization with p80 DNA binding site, even though the intact site binds DNA (Fig. 6). However, in agreement with previous gel mobility shift independently of p80. data (9, 10), neither free p70 nor the 430–609 mutant bound DISCUSSION DNA in the gel shift assay (Fig. 7). Moreover, dimerization of the 430–609 mutant with p80 did not restore DNA binding Binding of Ku to DNA is a critical step in the targeting and activation of DNA-dependent protein kinase (1–3). However, activity. the mechanism of DNA binding by Ku has not been defined DNA binding activity in the gel shift assay also depended on completely. In this study, two regions of p70 mediating dimer- a second site localized to amino acids 1–542 of p70 (Fig. 6). ization with p80 were defined, each of which is located in close Unlike the C-terminal DNA binding domain, activity of this proximity to one of the two DNA binding sites (Fig. 8). The N-terminal DNA binding domain required dimerization with existence of multiple dimerization and DNA binding sites may p80 via the N-terminal subunit interaction domain (Fig. 6). A explain some of the previous discrepancies regarding the inter- shorter fragment (p70 amino acids 1–115) also appeared to action of Ku with DNA. The C-terminal DNA binding domain of have weaker, p80-dependent binding activity. Interestingly, p70 corresponds to the minimal domain mapped previously (7), although similar in its requirement for p80 binding, the p70 whereas the N-terminal, p80-dependent domain may explain (1–542)-p80 dimer failed to bind DNA in the gel shift assay the p80 dependence of DNA binding in gel shift assays (9, 10). (Fig. 7) despite significant binding in the DNA immunoprecipi- Mechanism of p70-p80 Dimerization—One of the main find- tation assay (Fig. 6). The present studies have not addressed ings of the present study was that p70 has two dimerization the question of how DNA interacts with this domain. One sites. One site, localized to amino acids 430–482, is contained possibility is that both p70 and p80 participate. Alternatively, within the C-terminal 20-kDa portion of p70, a region impli- the interaction with DNA could be mediated by a conformation cated previously in subunit interaction using the yeast 2-hy- of p70 that depends on the binding of p80 or vice versa. Further brid system (10). This region does not contain the leucine studies will be needed to address this issue. zipper-like motif near the C-terminal end of p70 (amino acids We conclude that Ku antigen has at least two domains in- 483–504), consistent with the previous observation that a volved in DNA binding. One of these, the C-terminal portion of leucine zipper-like motif of p80 is not required for interaction p70, does not require interactions with p80. The other forms with p70 (10). The C-terminal interaction domain of p70 is upon dimerization of p70 (amino acids 1–542) with p80. Nei- related to the epitope recognized by mAb 162 (Figs. 3–4), which ther domain by itself is sufficient for DNA binding activity in is specific for the p70/p80 dimer and stabilizes the dimer under gel mobility shift assays, but together they confer activity. dissociating conditions (16). The p70 sequence recognized by Interestingly, although deletion of p70 amino acids 601–609 mAb 162 (amino acids 430–542) and the dimerization domain abrogates p80-independent DNA binding activity of the C-ter- mapped using recombinant baculovirus-expressed proteins minal DNA binding site, this activity apparently can be re- (amino acids 430–482) correlate well. Limited protease diges- stored by dimerization with full-length p80, since the p70 (1– tion confirmed the importance of this region in dimerization in 600)-p80 complex was active in the gel shift assay (Fig. 7). mammalian cells. The discrepancy between the gel mobility shift assay and More recently, autoantibodies were identified in autoim- DNA immunoprecipitation may reflect the ability of the latter 848 Functional Domains of Ku Antigen assay to detect lower affinity interactions between Ku and Acknowledgment—We thank Dr. Mark Knuth (Promega Inc. DNA than detected by the gel shift technique. Alternatively, Madison, WI) for supplying mAb N3H10 and Dr. Beverly Mitchell (University of North Carolina, Chapel Hill, NC) for critically reading the conditions used for the gel shift assay may induce confor- the manuscript. mational changes that alter the binding of free p70 to DNA. The DNA binding of both full-length p70 and the C-terminal REFERENCES portion of p70 clearly is conformation-sensitive since a pro- 1. Reeves, W. H., Wang, J., Ajmani, A. K., Stojanov, L., and Satoh, M. (1997) in longed renaturation step is required after transfer of the dena- The Antibodies (Zanetti, M., and Capra, J. D., eds), pp. 33–84, Harwood tured proteins to nitrocellulose membranes to demonstrate Academic Publishers, Amsterdam 2. Anderson, C. W. (1993) Trends Biochem. Sci. 18, 433–437 binding in Southwestern blot assays (6, 7). Finally, it is possi- 3. Weaver, D. T. (1995) Adv. Immunol. 58, 29–85 ble that free p70 is masked by other proteins or nucleic acids in 4. Griffin, W., Torrance, H., Rodda, D. J., Prefontaine, G. G., Pope, L., and Hache, the crude extracts utilized for gel shift assays. In contrast, p70 R. J. G. (1996) Nature 380, 265–268 5. Mimori, T., and Hardin, J. A. (1986) J. Biol. Chem. 261, 10375–10379 is affinity purified onto beads in the DNA immunoprecipitation 6. Allaway, G. P., Vivino, A. A., Kohn, L. D., Notkins, A. L., and Prabhakar, B. S. assay, and the high salt pre-washing of the beads prior to DNA (1989) Biochem. Biophys. Res. Commun. 168, 747–755 binding could release these inhibitory factors. 7. Chou, C. H., Wang, J., Knuth, M. W., and Reeves, W. H. (1992) J. Exp. Med. 175, 1677–1684 Functional Significance—The complexity of the interaction 8. Wang, J., Satoh, M., Chou, C. H., and Reeves, W. H. (1994) FEBS Lett. 351, between p70 and p80 and between Ku and DNA may be im- 219–224 9. Griffith, A. J., Blier, P. R., Mimori, T., and Hardin, J. A. (1992) J. Biol. Chem. portant for the various functions ascribed to Ku antigen. In 267, 331–338 addition to its role in end binding during DNA double strand 10. Wu, X. and Lieber, M. R. (1996) Mol. Cell. Biol. 16, 5186–5193 break repair and V(D)J recombination (22, 23), Ku antigen has 11. Wang, J., Dong, X., Stojanov, L., Kimpel, D., Satoh, M., and Reeves, W. H. (1997) Arthritis Rheum. 40, 1344–1353 sequence-specific DNA binding activity and may be involved in 12. Danska, J. S., Holland, D. P., Mariathasan, S., Williams, K. M., and Guidos, transcriptional activation (4) or repression (24, 25). The N- C. J. (1996) Mol. Cell. Biol. 16, 5507–5517 terminal acidic domain of p70 identified by sequence analysis 13. Han, Z., Johnston, C., Reeves, W. H., Carter, T., Wyche, J. H., and Hendrickson, E. A. (1996) J. Biol. Chem. 271, 14098–14104 (15) has strong transcriptional activation activity (10) although 14. Ono, M., Tucker, P. W., and Capra, J. D. (1994) Nucleic Acids Res. 22, Ku also may repress transcription (24, 25). 3918–3924 It remains unclear whether the portions of Ku mediating end 15. Reeves, W. H., and Sthoeger, Z. M. (1989) J. Biol. Chem. 264, 5047–5052 16. Wang, J., Satoh, M., Pierani, A., Schmitt, J., Chou, C. H., Stunnenberg, H. G., binding activity are the same as those responsible for sequence- Roeder, R. G., and Reeves, W. H. (1994) J. Cell Sci. 107, 3223–3233 specific binding. Previous studies suggest that the C-terminal 17. Davidson, H. W. and Watts, C. (1989) J. Cell Biol. 109, 85–92 DNA binding domain of p70 binds preferentially to DNA ends 18. Schwyzer, M., Weil, R., Frank, G., and Zuber, H. (1980) J. Biol. Chem. 255, 5627–5634 (8). However, we have not yet examined whether the p80-de- 19. de Vries, E., van Driel, W., Bergsma, W. G., Arnberg, A. C., and van der Vliet, pendent DNA binding site identified here displays a similar P. C. (1989) J. Mol. Biol. 208, 65–78 preference for ends, nor have we investigated the role of the 20. Romero, F., Dargemont, C., Pozo, F., Reeves, W. H., Camonis, J., Gisselbrecht, S., and Fischer, S. (1996) Mol. Cell. Biol. 16, 37–44 two DNA binding sites in sequence specific DNA binding to the 21. Hartley, K. O., Gell, D., Smith, G. C. M., Zhang, H., Divecha, N., Connelly, M., LTR sequence of mouse mammary tumor virus (4). The answer Admon, A., Lees-Miller, S. P., Anderson, C. W., and Jackson, S. (1995) Cell to these questions may help to further define the mechanisms 82, 849–856 22. Smider, V., Rathmell, W. K., Lieber, M. R., and Chu, G. (1994) Science 266, by which Ku autoantigen functions in DNA repair and tran- 288–291 scriptional activation/repression. Ku antigen may be one of a 23. Taccioli, G. E., Gottlieb, T. M., Blunt, T., Priestley, A., Demengeot, J., Mizuta, R., Lehmann, A. R., Alt, F. W., Jackson, S. P., and Jeggo, P. A. (1994) number of multifunctional DNA binding proteins involved in Science 265, 1442–1445 transcription as well as other aspects of nucleic acid metabo- 24. Kuhn, A., Gottlieb, T. M., Jackson, S. P., and Grummt, I. (1995) Genes Dev. 9, lism, such as DNA synthesis or repair (26). These distinct 193–203 25. Kuhn, A., Stefanovsky, V., and Grummt, I. (1993) Nucleic Acids Res. 21, functions potentially could be mediated by different DNA bind- 2057–2063 ing sites or interaction states of the p70 and p80 subunits. 26. DePamphilis, M. L. (1988) Cell 52, 635–638