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A Model for Ku Heterodimer Assembly and Interaction with DNA

1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês

10.1074/jbc.273.47.31068

1083-351X

Jingsong Wang, Xingwen Dong, Westley H. Reeves,

Genomics and Chromatin Dynamics

Ku autoantigen, a heterodimer of 70- and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair. Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion. The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent. Both must be intact for end binding activity in gel shift assays. To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses. We show by several criteria that amino acids 371–510 of p80 interact with p70. Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region. In DNA immunoprecipitation assays, amino acids 179–510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179–732 were necessary. Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases. Ku autoantigen, a heterodimer of 70- and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair. Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion. The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent. Both must be intact for end binding activity in gel shift assays. To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses. We show by several criteria that amino acids 371–510 of p80 interact with p70. Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region. In DNA immunoprecipitation assays, amino acids 179–510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179–732 were necessary. Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases. double-stranded double strand break repair polyacrylamide gel electrophoresis monoclonal antibody x-ray cross-complementing. Ku antigen was identified and characterized using autoantibodies from the sera of patients with systemic autoimmune diseases (reviewed in Ref. 1Reeves W.H. Wang J. Ajmani A.K. Stojanov L. Satoh M. Zanetti M. Capra J.D. The Antibodies. Harwood Academic Publishers, Amsterdam1997: 33-84Google Scholar). Later, it was shown to be associated with a DNA-dependent protein kinase that phosphorylates chromatin-bound proteins in vitro (2Anderson C.W. Lees-Miller S.P. CRC Crit. Rev. Eukaryotic Gene Exp. 1992; 2: 283-314PubMed Google Scholar) and is involved in double-stranded (ds)1 DNA break repair (DSBR), V(D)J recombination, and isotype switching (3Weaver D.T. CRC Crit. Rev. Eukaryotic Gene Exp. 1996; 6: 345-375Crossref PubMed Scopus (44) Google Scholar, 4Chu G. J. Biol. Chem. 1997; 272: 24097-24100Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 5Casellas R. Nussenzweig A. Wuerffel R. Pelanda R. Reichlin A. Suh H. Qin X.F. Besmer E. Kenter A. Rajewsky K. Nussenzweig M.C. EMBO J. 1998; 17: 2404-2411Crossref PubMed Scopus (292) Google Scholar), as well as telomeric length maintenance and silencing (6Boulton S.J. Jackson S.P. EMBO J. 1998; 17: 1819-1828Crossref PubMed Scopus (554) Google Scholar). Ku is a heterodimer of 70-kDa (p70) and ∼80-kDa (p80) subunits that binds dsDNA ends (1Reeves W.H. Wang J. Ajmani A.K. Stojanov L. Satoh M. Zanetti M. Capra J.D. The Antibodies. Harwood Academic Publishers, Amsterdam1997: 33-84Google Scholar, 7Mimori T. Hardin J.A. J. Biol. Chem. 1986; 261: 10375-10379Abstract Full Text PDF PubMed Google Scholar). Sequence-specific DNA binding also has been reported (1Reeves W.H. Wang J. Ajmani A.K. Stojanov L. Satoh M. Zanetti M. Capra J.D. The Antibodies. Harwood Academic Publishers, Amsterdam1997: 33-84Google Scholar, 8Griffin W. Torrance H. Rodda D.J. Prefontaine G.G. Pope L. Hache R.J.G. Nature. 1996; 380: 265-268Crossref PubMed Scopus (197) Google Scholar). Defining the contribution of p70 and p80 to DNA binding is of interest because of the dual sequence- and end-specific binding of Ku and because of uncertainty as to its precise role in DNA repair. In DNA immunoprecipitation and Southwestern blot assays, p70 binds DNA in the absence of p80 (7Mimori T. Hardin J.A. J. Biol. Chem. 1986; 261: 10375-10379Abstract Full Text PDF PubMed Google Scholar, 9Allaway G.P. Vivino A.A. Kohn L.D. Notkins A.L. Prabhakar B.S. Biochem. Biophys. Res. Commun. 1989; 168: 747-755Crossref Scopus (33) Google Scholar, 10Chou C.H. Wang J. Knuth M.W. Reeves W.H. J. Exp. Med. 1992; 175: 1677-1684Crossref PubMed Scopus (67) Google Scholar), but in gel shift assays, only the dimer can bind (11Griffith A.J. Blier P.R. Mimori T. Hardin J.A. J. Biol. Chem. 1992; 267: 331-338Abstract Full Text PDF PubMed Google Scholar, 12Wu X. Lieber M.R. Mol. Cell. Biol. 1996; 16: 5186-5193Crossref PubMed Scopus (110) Google Scholar). Although p80 alone does not bind to DNA, DSBR mutants in the XRCC5 complementation group have defects in the p80 gene (13Errami A. Smider V. Rathmell W.K. He D.M. Hendrickson E.A. Zdzienicka M.Z. Chu G. Mol. Cell. Biol. 1996; 16: 1519-1526Crossref PubMed Scopus (158) Google Scholar, 14Singleton 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, 15Jin S. Weaver D.T. EMBO J. 1997; 16: 6874-6885Crossref PubMed Scopus (125) Google Scholar). Defining the mechanism of p70-p80 dimerization may help to explain the role of p80 in sequence-specific DNA binding, DNA end binding, and/or DSBR. The p70 subunit contains two p80 interaction domains, amino acids 1–115 and 430–482, respectively, as well as two regions involved in DNA binding, each partially overlapping one of the interaction domains (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). A p80-independent DNA binding site is located on the C terminus (amino acids 536–609) (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 17Wang J. Satoh M. Chou C.H. Reeves W.H. FEBS Lett. 1994; 351: 219-224Crossref PubMed Scopus (26) Google Scholar), whereas the N-terminal region must bind p80 in order to bind DNA. The goal of this study was to define the dimerization-dependent DNA binding site. Using p70 and p80 mutants, a large central domain of p80 involved in both dimerization and DNA binding was identified. This site, like the p80-independent DNA binding site, preferentially recognized DNA ends, suggesting that a single Ku heterodimer may bind two DNA ends. K562 (human erythroleukemia) cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/streptomycin. The Sf9 (Spodoptera frugiperda ovary) cell line was obtained from the ATCC, and maintained at 27 °C in Grace's insect tissue culture medium supplemented with 3.3 g/liter TC yeastolate, 3.3 g/liter lactalbumin hydrolyzate, 10% fetal bovine serum, and penicillin/streptomycin. The human p70 and p80 Ku cDNAs were subcloned into a modified baculovirus transfer vector pVLB4-mp53 and co-transfected with linearized wild-type baculovirus into Sf9 cells as described (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The recombinant baculovirus vectors direct the expression of Ku proteins fused to an N-terminal polyhistidine sequence and a enterokinase cleavage site. The recombinant baculoviruses expressing the full-length p80 or p70 are designated p80-bv and p70-bv respectively. Recombinant baculoviruses expressing mutant human p80 were constructed in the same manner. Truncated p80 cDNAs were amplified by polymerase chain reaction using primer pairs listed in Table I, and subcloned into pVLB4-mp53.Table IPrimers used to amplify Ku cDNAs for subcloning into baculovirus transfer vectorsConstructs5′ Primer3′ Primerp80.1–1785′-GCGGATCCATGGTGCGGTCGGGGAATAAG-3′5′-GCGAATTCTCATCTGTCCCCACTTCCATC-3′p80.371–7325′-GCGGATCCGAGGCAGCTGCAGTTGCAC-3′5′-GCGAATTCCGACCTATATCATGTCC-3′p80.179–7325′-GCGGATCCGGAGATGGCCCCTTTCGC-3′5′-GCGAATTCCGACCTATATCATGTCC-3′p80.179–6545′-GCGGATCCGGAGATGGCCCCTTTCGC-3′5′-GCGAATTCTCAGCGCTGCTCTTCTGAAAAC-3′p80.179–5105′-GCGGATCCGGAGATGGCCCCTTTCGC-3′5′-GCGAATTCTCACTGCTGAATTGGGGGTAG-3′p80.179–4005′-GCGGATCCGGAGATGGCCCCTTTCGC-3′5′-GCGAATTCTCATCTTTTGTCATAAGC-3′p80.371–6545′-GCGGATCCGAGGCAGCTGCAGTTGCAC-3′5′-GCGAATTCTCAGCGCTGCTCTTCTGAAAAC-3′p80.371–5105′-GCGGATCCGAGGCAGCTGCAGTTGCAC-3′5′-GCGAATTCTCACTGCTGAATTGGGGGTAG-3′p80.400–7325′-GCGGATCCAGAGCTAATCCTCAAGTC-3′5′-GCGAATTCCGACCTATATCATGTCC-3′p80.400–6545′-GCGGATCCAGAGCTAATCCTCAAGTC-3′5′-GCGAATTCTCAGCGCTGCTCTTCTGAAAAC-3′p80.400–5105′-GCGGATCCAGAGCTAATCCTCAAGTC-3′5′-GCGAATTCTCACTGCTGAATTGGGGGTAG-3′p80.510–6545′-GCGGATCCCAGCATATTTGGAATATGC-3′5′-GCGAATTCTCAGCGCTGCTCTTCTGAAAAC-3′p80.654–7325′-GCGGATCCCGCTTTAACAACTTCCTG-3′5′-GCGAATTCCGACCTATATCATGTCC-3′ Open table in a new tab Sf9 cells were infected with one or more recombinant baculoviruses as described (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Seventy-two hours after infection, recombinant Ku proteins were identified in cell lysates by SDS-PAGE and immunoblotting using human autoimmune serum and/or specific monoclonal antibodies (mAbs). mAbs specific for the human Ku antigen were as described previously (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Their isotypes and specificities are as follows: 162, IgG2a anti-p70/p80 dimer; S10B1, IgG1 anti- p80 (amino acids 179–371); 111, IgG1 anti-p80 (amino acids 610–705); N3H10, IgG2b anti-p70 (amino acids 529–541). An IgG2a murine mAb specific for the polyhistidine tag (HIS-1) was from Sigma. Immunoblot analysis of the recombinant p70 and p80 proteins from Sf9 cells or Ku proteins from K562 cells was performed using murine mAbs (1:1000 dilution) (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Radiolabeling of baculovirus-infected Sf9 cells with [35S]methionine/cysteine and immunoprecipitation onto protein A-Sepharose beads were carried out as described (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The beads were washed with 1.5 m NaCl NET-Nonidet P-40 buffer (1.5 m NaCl, 50 mm Tris, pH 7.5, 0.3% Nonidet P-40, 2 mm EDTA) and once with NET buffer (0.15m NaCl, 50 mm Tris, pH 7.5, 2 mmEDTA). Immunoprecipitated proteins were analyzed by SDS-PAGE. Ku heterodimer from K562 cells was affinity-purified onto mAb 162-coated protein A-Sepharose beads and digested with proteases (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Protease V8 (Boehringer Mannheim), trypsin-treated tosylphenylalanyl chloromethyl ketone (Millipore Corp., Freehold, NJ), or chymotrypsin (Worthington Biochemical Corp., Freehold, NJ) were added to the beads at 2–200 μg/ml in 10 mm Tris-HCl, pH 7.5, and 2.5 mmCaCl2 for 30 min at 22 °C. The beads were washed, and p80 fragments remaining associated with the beads were identified by SDS-PAGE and immunoblotting using S10B1. Sf9 or K562 cells were washed briefly with hypotonic buffer (10 mm HEPES, pH 7.3, 25 mm KCl, 10 mm NaCl, 1 mmMgCl2, 0.1 mm EDTA), and pellets were resuspended at 2 × 108 cells/ml in the same buffer supplemented with 0.5 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, and aprotinin (0.3 trypsin inhibitor units/ml). Pellets were lysed by three freeze-thaw cycles and adjusted to 0.5 m KCl and 10 mm MgCl2. After centrifuging, the supernatant was diluted to 0.1 m KCl and stored at −80 °C. A 564-base pair linear double-stranded DNA fragment from bacteriophage λ was radiolabeled with [32P]dATP, and its binding to affinity-purified Ku antigen was measured by DNA immunoprecipitation assay (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, cell extract was immunoprecipitated on protein A-Sepharose beads with 3 μl of N3H10, 111, or HIS-1 ascitic fluid. Equal amounts of recombinant Ku proteins were bound to the beads, as determined by SDS-PAGE with Coomassie Blue staining. Beads were washed with 1.5m NaCl NET-Nonidet P-40 buffer, followed by the same buffer containing 50 mm NaCl. Radiolabeled DNA (25 ng) was added for 1 h, and the beads were washed again with NET-Nonidet P-40. Bound DNA was recovered by proteinase K digestion, and an aliquot of the supernatant was used for scintillation counting. For gel mobility shift assays, radiolabeled DNA was purified using a Geneclean III kit (Bio 101, Vista, CA) and then incubated with cell extracts in 20 μl of binding buffer (10 mm Tris, pH 7.5, 1 mm dithiothreitol, 5 mm EDTA, 10% glycerol, 150 mm NaCl, 2 μg of closed circular φX174 Rf I DNA) at 22 °C for 30 min (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Samples were analyzed by electrophoresis in a 4% polyacrylamide gel, followed by autoradiography. For supershift experiments, purified mAbs (∼1 μg) were added to the binding mixture 5 min after adding probe and incubated for an additional 30 min at 22 °C before electrophoresis. Two regions of the p70 interact with p80. Binding of p80 to one of these interaction domains contributes to the DNA binding activity of Ku. In the present study, we investigated the mechanism of p80-p70 dimerization and its role in DNA binding. The leucine zipper-like sequence of p70 is not required for dimerization with p80 (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The role of the leucine zipper-like sequence near the N terminus of p80 (18Yaneva M. Wen J. Ayala A. Cook R. J. Biol. Chem. 1989; 264: 13407-13411Abstract Full Text PDF PubMed Google Scholar) in interactions with p70 was investigated by co-expressing a panel of p80 deletion mutants (Fig. 1 A) with full-length p70 in Sf9 cells using recombinant baculoviruses. Recombinant protein expression was verified by immunoprecipitation using the polyhistidine-specific mAb HIS-1 (Fig. 1,B and C, HIS). To detect heterodimer formation, the p80 mutants were co-immunoprecipitated with anti-p70 mAb N3H10 (Fig. 1, B and C, N3H10). Mutant proteins 179–732, 179–654, 179–510, 371–654, and 371–510 were co-immunoprecipitated by N3H10, but 179–400 was not (Fig. 1 B), suggesting that an interaction domain is located in the central portion of p80 (amino acids 371–510), and that the leucine zipper-like region (amino acids 146–178) is not essential for dimerization with p70. Even though 179–654 and 179–510 dimerized with p70 less efficiently than 179–732, their interactions were visualized readily by co-immunoprecipitation assay. Since 179–510 interacted with p70, whereas 179–400 did not, amino acids 400–510 may be crucial for dimerization with p70. However, because 179–400 was expressed less efficiently than the other constructs, it is difficult to completely exclude the possibility that some dimerization might occur with this construct. Mutant 1–178 overlaps the leucine zipper-like sequence, but was not co-immunoprecipitated by N3H10 (Fig. 1 C), indicating that the leucine zipper-like sequence of p80, like that of p70, is not required for dimerization. Mutant 400–732 was co-immunoprecipitated by N3H10 but not 400–510, 510–654, or 654–732 (Fig. 1 C). Thus, even though amino acids 400–510 are crucial for p70-p80 dimerization, this region by itself is insufficient. Additional amino acids either N-terminal (e.g. mutant 371–510) or C-terminal (e.g. mutant 400–732) to the "core" domain (amino acids 400–510) are necessary for binding to p70. mAb 162 recognizes an epitope created by dimerization of p70 with p80 and prevents their dissociation in the presence of 1.5 m NaCl and detergent (19Wang J. Satoh M. Pierani A. Schmitt J. Chou C.H. Stunnenberg H.G. Roeder R.G. Reeves W.H. J. Cell Sci. 1994; 107: 3223-3233PubMed Google Scholar). The 162 binding site on p70 (amino acids 430–542) overlaps the C-terminal interaction domain of p70 (amino acids 430–482). The relationship of the 162 binding site to the interaction domain of p80 was investigated (Fig. 2,A–C). Expression of p70 and p80 deletion mutants was verified by HIS-1 antibody. mAb 162 immunoprecipitated full-length p70 when co-expressed with p80 mutant 179–732, but not 371–732, 179–371, or 1–178 (Fig. 2 B), suggesting that the C-terminal half of p80 contributes to formation of the epitope. mAb 162 also immunoprecipitated p70 when co-expressed with p80 179–654 or 179–510, but not 179–400 (Fig. 2 C), but it did not immunoprecipitate p70 or any of the p80 deletion mutants when expressed alone in Sf9 cells (20Wang J. Dong X. Stojanov L. Kimpel D. Satoh M. Reeves W.H. Arthritis Rheum. 1997; 40: 1344-1353Crossref PubMed Scopus (10) Google Scholar). These data confirm the dimerization studies illustrated in Fig. 1 B. We conclude that amino acids 179–510 of p80 contribute to formation of the 162 epitope, and that this region overlaps the dimerization domain of p80. To further define the minimal 162 epitope, p70 (amino acids 430–542) (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) was co-expressed with p80 mutants. mAb 162 immunoprecipitated p70 (430–542) when co-expressed with p80 (179–654 or 179–510), but not p80 (179–400) (data not shown), indicating that the 162 epitope consists of p70 (430–542) plus p80 (179–510). To confirm the co-immunoprecipitation data, dimerization of p70 and p80 was studied in the human K562 cell line. Binding of mAb 162 protects the p70 interaction domain from proteolysis (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), suggesting that the corresponding region of p80 may be protected, as well. Ku heterodimer affinity-purified on mAb 162 was treated with proteases, and p80 fragments retained on 162 beads after protease treatment were analyzed by Western blotting using mAb S10B1 (Fig. 3). The smallest bead-associated p80 fragment after chymotrypsin digestion was a weak, but consistently visualized, band migrating at ∼22 kDa. Western blot analysis using mAb 111 (anti-p80, 610–705) failed to detect fragments smaller than 60 kDa (data not shown). Since S10B1 recognizes amino acids 179–371 of p80, the protease studies support the immunoprecipitation data, indicating that a dimerization site is located in the central portion of p80. The p70 protein carries two separate domains (amino acids 1–115 and 430–482) (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) that apparently interact with a single domain of p80 (amino acids 371–510). Heterodimer assembly was evaluated further by co-expressing p70 deletion mutants overlapping the two dimerization domains separately or together with p80, and immunoprecipitating with anti-Ku mAbs (Fig. 4). Protein expression was verified by anti-polyhistidine immunoprecipitation (Fig. 4, HIS). When co-expressed individually with p80, p70 (1–433) as well as p70 (430–609) could be co-immunoprecipitated by anti-p80 mAb 111 (Fig. 4, 111). In addition, both 1–433 and 430–609 were co-immunoprecipitated by mAb 111 when co-expressed together with p80. The bands intensities were similar regardless of whether the p70 proteins were co-expressed with p80 separately (1–433, 430–609) or together (1–433 + 430–609), suggesting that the p70 mutants do not interfere with each other's binding to p80. Anti-p70 mAb N3H10 also co-immunoprecipitated p80 in 430–609/p80 co-expressing lysates. More importantly, N3H10 co-immunoprecipitated 1–433 along with p80. Some free 430–609 also was immunoprecipitated, accounting for the greater intensity of that band (Fig. 4, right panel). N3H10 does not immunoprecipitate 1–433 when the mutant protein is expressed alone or co-expressed with 430–609 (data not shown), indicating that the two p70 dimerization domains can bind independently to the same p80 molecule. Similar results were obtained when smaller fragments of p70 and p80 carrying dimerization sites were investigated in the same manner (data not shown). Taken together, these data indicate that the two dimerization domains of p70 interact with the central region of p80 and do not compete with one another for binding. The p70 protein has two DNA binding domains, each partially overlapping one of the dimerization sites. The C-terminal site (amino acids 536–609) has DNA end binding activity by itself, whereas N-terminal DNA binding activity requires dimerization with p80. Partial activity is exhibited by amino acids 1–115, and complete activity by amino acids 1–542. Thus, p70 deletion mutants lacking the p80 independent C-terminal domain, such as 1–600, 1–542, or 1–115, bind DNA in a p80-dependent manner. To determine how the p80-dependent site is assembled, the DNA binding activity of p70 (1–600) co-expressed with a panel of p80 mutants overlapping the dimerization domain of p80 was tested (Fig. 5). It was necessary to use DNA immunoprecipitation assays because binding in gel shift assays requires both DNA binding sites (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Consistent with previous findings, wild-type p70 by itself bound to linear dsDNA as well as did the p70/p80 heterodimer (Fig. 5, left). The p70 deletion mutant 1–600 bound DNA poorly, but clearly better than extract from cells infected with wild-type (WT) baculovirus. However, its activity was enhanced considerably by dimerization with p80. Partial p80-dependent DNA binding activity was conferred when p70 (1–600) dimerized with the p80 deletion mutants 179–732, 179–654, or 179–510, each of which overlaps the interaction domain of p80. Decreased binding of the mutants may relate to differences in dimerization efficiency. However, comparable amounts of Ku dimers, determined in preliminary co-immunoprecipitation experiments using N3H10, were used in each lane, arguing that the dimers containing truncated p80 constructs may bind DNA less well. We conclude that p80 (179–510) mediates the p80-dependent DNA binding activity of p70. End binding activity of Ku in gel shift assays requires both the N- and C-terminal DNA binding domains of p70 (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Because p80 is needed for activity of the N-terminal domain, it also is required for binding in gel shift assays (11Griffith A.J. Blier P.R. Mimori T. Hardin J.A. J. Biol. Chem. 1992; 267: 331-338Abstract Full Text PDF PubMed Google Scholar, 12Wu X. Lieber M.R. Mol. Cell. Biol. 1996; 16: 5186-5193Crossref PubMed Scopus (110) Google Scholar). To establish the region of p80 conferring DNA binding activity in gel shift assays, the DNA binding of p80 deletion mutants co-expressed with wild-type p70 was tested. Consistent with previous findings, recombinant Ku heterodimer bound DNA efficiently, whereas p70, p80, and p80 deletion mutants by themselves failed to bind in the gel shift assay (Fig. 6 and data not shown). The p80 mutant 179–732, but not 371–732, 179–654, 179–510, or 179–400, bound DNA after dimerizing with wild-type p70 (Fig. 6). The multiple bands visible on the gel (Fig. 6, 179–732/p70 lane, B1, B2, B3) suggest that dimerization also restores the internal translocation property of Ku (21de Vries E. van Driel W. Bergsma W.G. Arnberg A.C. van der Vliet P.C. J. Mol. Biol. 1989; 208: 65-78Crossref PubMed Scopus (216) Google Scholar). Binding of the recombinant protein 179–732/p70 to DNA was verified by supershift assays using anti-Ku mAb 162 and anti-polyhistidine mAb HIS-1 (data not shown). Paradoxically, we have shown previously that dimerization with wild-type p80 compensates partially for the deletion of a portion (amino acids 601–609) of the p70 C-terminal DNA binding domain (p70 (1–600), p70Δ), even though the intact C-terminal domain binds DNA independently of p80 (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, p80 (179–732) failed to restore the DNA binding activity of p70Δ (Fig. 6, 179–732/p70Δ lane). Thus, p80 (179–732) was required for Ku DNA end binding activity in the gel shift assay, but this region was insufficient to restore the DNA binding activity of p70Δ. Only full-length p80 exhibited the latter effect. The C-terminal DNA binding domain of p70 recognizes dsDNA termini preferentially (17Wang J. Satoh M. Chou C.H. Reeves W.H. FEBS Lett. 1994; 351: 219-224Crossref PubMed Scopus (26) Google Scholar), and could, by itself, explain the end binding activity of Ku antigen. The role of the p80-dependent DNA binding site has not been defined. To evaluate whether this site also exhibits preferential end binding activity, competition experiments were carried out as detailed in Ref.17Wang J. Satoh M. Chou C.H. Reeves W.H. FEBS Lett. 1994; 351: 219-224Crossref PubMed Scopus (26) Google Scholar. Consistent with previous findings, binding of the intact p70/p80 heterodimer to a linear double-stranded DNA probe was preferentially inhibited by linear dsDNA (Fig. 7). There was little inhibition of binding by closed circular DNA (φX174 Rf I DNA or pBKS plasmid DNA) or by tRNA. The full-length p70 protein by itself and p70 (amino acids 430–609) displayed a similar inhibition pattern, suggesting that p70 also binds preferentially to DNA termini. Dimerization of each of the p70 DNA binding domains (p70.430–609 and p70.1–542, respectively) with full-length p80 resulted in constructs that also bound preferentially to linear dsDNA. Binding was not inhibited significantly by φX174 Rf I DNA, pBKS plasmid DNA, or tRNA. Deficiency of Ku autoantigen leads to defective DSBR and V(D)J recombination, resulting in x-ray sensitivity and severe combined immunodeficiency (22Weaver D.T. Adv. Immunol. 1995; 58: 29-85Crossref PubMed Scopus (59) Google Scholar). Despite the fact that the p70 Ku subunit can bind dsDNA ends independently of p80 (17Wang J. Satoh M. Chou C.H. Reeves W.H. FEBS Lett. 1994; 351: 219-224Crossref PubMed Scopus (26) Google Scholar), DSBR is absent in cells with p80 mutations (13Errami A. Smider V. Rathmell W.K. He D.M. Hendrickson E.A. Zdzienicka M.Z. Chu G. Mol. Cell. Biol. 1996; 16: 1519-1526Crossref PubMed Scopus (158) Google Scholar, 14Singleton 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), apparently due to the existence of a second, dimerization-dependent, DNA binding site (16Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In the present study, amino acids 371–510 of p80 was shown to mediate dimerization with p70 and recognition by the Ku dimer-specific mAb 162, in addition to participating in DNA binding. As illustrated in Fig. 8, the locations of the DNA binding and dimerization domains of p80 are consistent with the DSBR-deficient phenotypes of the XR-V9B and XR-V15B cell lines (13Errami A. Smider V. Rathmell W.K. He D.M. Hendrickson E.A. Zdzienicka M.Z. Chu G. Mol. Cell. 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