摘要
Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+ in vitro. Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD+. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activation in vivo as well. Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+ in vitro. Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD+. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activation in vivo as well. poly(ADP-ribose) polymerase-1 tumor necrosis factor-α interleukin-1 electrophoretic mobility shift assay 3-aminobenzamide phenylmethylsulfonyl fluoride leucine zipper immediate upstream region Protein poly(ADP-ribosyl)ation is a post-translational modification of DNA binding proteins in eucaryotes in vivo(1de Murcia G. Menissier de Murcia J. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (766) Google Scholar, 2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). The acute synthesis of poly(ADP-ribose) from βNAD+ in response to DNA strand break formation is mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1)1 (1de Murcia G. Menissier de Murcia J. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (766) Google Scholar, 2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). PARP-1 is an abundant constitutively expressed nuclear enzyme (3Yamanaka H. Penning C.A. Willis E.H. Wasson D.B. Carson D.A. J. Biol. Chem. 1988; 263: 3879-3883Abstract Full Text PDF PubMed Google Scholar, 4Ludwig A. Behnke B. Holtlund J. Hilz H. J. Biol. Chem. 1988; 263: 6993-6999Abstract Full Text PDF PubMed Google Scholar). It is also a phylogenetically ancient protein widely conserved in eucaryotes, with a noticeable exception in yeast (2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). The modular structure of PARP-1 indicates that this protein contains bipartite zinc fingers (5Mazen A. Menissier-de Murcia J. Molinete M. Simonin F. Gradwohl G. Poirier G. de Murcia G. Nucleic Acids Res. 1989; 17: 4689-4698Crossref PubMed Scopus (82) Google Scholar) in its N-terminal sequence. When bound to DNA nicks, the zinc finger motifs activate the C-terminal catalytic domain of PARP-1 to processively transfer the ADP-ribose moiety from βNAD+ to covalently modify acceptor proteins (5Mazen A. Menissier-de Murcia J. Molinete M. Simonin F. Gradwohl G. Poirier G. de Murcia G. Nucleic Acids Res. 1989; 17: 4689-4698Crossref PubMed Scopus (82) Google Scholar, 6Mendoza-Alvarez H. Alvarez-Gonzalez R. J. Biol. Chem. 1993; 268: 22575-22580Abstract Full Text PDF PubMed Google Scholar). This dynamic synthesis and rapid clearance (7Alvarez-Gonzalez R. Althaus F.R. Mutat. Res. 1989; 218: 67-74Crossref PubMed Scopus (187) Google Scholar) of protein-bound (ADP-ribose) polymers has been implicated in eucaryotic DNA repair, DNA replication, and transcription. During this process, numerous nuclear proteins, including histones, DNA polymerases and ligases, Ca2+/Mg2+-dependent endonuclease, and transcription factors (e.g. TFIIF, YY1, and p53) are covalently poly(ADP-ribosyl)ated in vitro and/orin vivo (8Tanaka Y. Yoshihara K. Itaya A. Kamiya T. Koide S.S. J. Biol. Chem. 1984; 259: 6579-6585Abstract Full Text PDF PubMed Google Scholar, 9Wesierska-Gadek J. Bugajska-Schretter A. Cerni C. J. Cell. Biochem. 1996; 62: 90-101Crossref PubMed Scopus (79) Google Scholar, 10Rawling J.M. Alvarez-Gonzalez R. Biochem. J. 1997; 324: 249-253Crossref PubMed Scopus (58) Google Scholar, 11Kumari S.R. Mendoza-Alvarez H. Alvarez-Gonzalez R. Cancer Res. 1998; 58: 5075-5078PubMed Google Scholar, 12Oei S.L. Griesenbeck J. Schweiger M. Ziegler M. J. Biol. Chem. 1998; 273: 31644-31647Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 13Yakovlev A.G. Wang G. Stoica B.A. Boulares H.A. Spoonde A.Y. Yoshihara K. Smulson M.E. J. Biol. Chem. 2000; 275: 21302-21308Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Furthermore, PARP-1 appears to also be involved in cellular commitment to apoptosis, because the proteolytic cleavage of PARP-1 by caspases 3 and/or 7 is frequently used as a hallmark of apoptotic execution (14Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2356) Google Scholar, 15Oliver F.J. Menissier-de Murcia J. de Murcia G. Am. J. Hum. Genet. 1999; 64: 1282-1288Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In fact, it has been suggested that the cleavage of PARP-1 allows cells to conserve energy reserves (βNAD+ and ATP) by inactivating the ADP-ribose polymerizing activity of PARP-1 (15Oliver F.J. Menissier-de Murcia J. de Murcia G. Am. J. Hum. Genet. 1999; 64: 1282-1288Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Until recently, most studies on PARP-1 had focused on its enzymatic activity following DNA damage (1de Murcia G. Menissier de Murcia J. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (766) Google Scholar, 2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). However, it has also been suggested that PARP-1 may play a more sophisticated molecular role in chromatin structure and function by forming protein complexes with other proteins. For example, PARP-1 physically associates with DNA polymerase α and stimulates DNA replication in vitrowithout degrading βNAD+ (16Simbulan C.M. Suzuki M. Izuta S. Sakurai T. Savoysky E. Kojima K. Miyahara K. Shizuta Y. Yoshida S. J. Biol. Chem. 1993; 268: 93-99Abstract Full Text PDF PubMed Google Scholar). In addition, PARP-1 enhances activator-dependent transcription as an active component of the pre-initiation complex in vitro, and this enhancement appears to be silenced by its auto-poly(ADP-ribosyl)ation (17Meisterernst M. Stelzer G. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2261-2265Crossref PubMed Scopus (145) Google Scholar). Roeder and co-workers (18Slattery E. Dignam J.D. Matsui T. Roeder R.G. J. Biol. Chem. 1983; 258: 5955-5959Abstract Full Text PDF PubMed Google Scholar) previously concluded that transcription factor TFIIC, a protein that stimulated nick translation, was identical to PARP-1. More recently, PARP-1 has also been shown to bind the oncogenic protein B-MYB to enhance its transactivating property (19Cervellera M.N. Sala A. J. Biol. Chem. 2000; 275: 10692-10696Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Therefore, PARP-1 may regulate the expression of specific genes by physical association with specific transcription factors. A good example for PARP-1-regulated gene expression events may be execution of the cell death program (15Oliver F.J. Menissier-de Murcia J. de Murcia G. Am. J. Hum. Genet. 1999; 64: 1282-1288Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Because the degradation of PARP-1 triggers the execution phase of apoptosis (14Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2356) Google Scholar) and NF-κB is considered an anti-apoptotic transcription factor, we hypothesized that the nuclear activation of NF-κB might also be regulated by PARP-1. Transcription factor NF-κB was originally described in B-lymphoid cells (20Sen R. Baltimore D. Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1958) Google Scholar). Classic NF-κB is a heterodimer composed of a DNA-binding p50 subunit and a transactivating p65-subunit (RelA). NF-κB is pre-synthesized in the cytosol and immediately sequestered as a protein complex with IκB in the cytoplasm (21Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1695) Google Scholar). NF-κB is activated for nuclear translocation by specific extracellular stimuli. In fact, this phenomenon has been shown to be independent of protein synthesis (22Sen R. Baltimore D. Cell. 1986; 47: 921-928Abstract Full Text PDF PubMed Scopus (1479) Google Scholar). A plethora of heterogeneous, seemingly unrelated signal molecules, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), lipopolysaccharide, γ-radiation, etoposide, or H2O2 can activate NF-κB in target cells. These stimuli activate a signal transduction cascade that targets IκB degradation in the cytoplasm. As a result of this process NF-κB reveals its nuclear localization signal and translocates to the nucleus. The rapidly nuclear-translocated NF-κB activates genes concerned with inflammatory or immune responses such as inducible nitric-oxide synthase, IL-1 β, IL-6, and TNF-α. Other studies have shown that NF-κB activation is accompanied by the intracellular generation of reactive oxygen species (23Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3436) Google Scholar, 24Schreck R. Albermann K. Baeuerle P.A. Free Radic. Res. Commun. 1992; 17: 221-237Crossref PubMed Scopus (1305) Google Scholar, 25Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1272) Google Scholar, 26Schmidt K.N. Traenckner E.B. Meier B. Baeuerle P.A. J. Biol. Chem. 1995; 270: 27136-27142Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). For example, the addition of micromolar concentrations of H2O2 can also activate NF-κB in Jurkat and HeLa cells, a process that may be blocked in the presence of antioxidants (23Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3436) Google Scholar, 25Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1272) Google Scholar). The notion that H2O2 may specifically lead to NF-κB nuclear targeting is noteworthy, because H2O2 can also activate the protein-poly(ADP-ribosyl)ation pathway and automodification reaction of PARP-1 by causing DNA strand breaks (27Schraufstatter I.U. Hinshaw D.B. Hyslop P.A. Spragg R.G. Cochrane C.G. J. Clin. Invest. 1986; 77: 1312-1320Crossref PubMed Scopus (437) Google Scholar, 28Schraufstatter I.U. Hyslop P.A. Hinshaw D.B. Spragg R.G. Sklar L.A. Cochrane C.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4908-4912Crossref PubMed Scopus (438) Google Scholar, 29Hyslop P.A. Hinshaw D.B. Halsey W.A. Schraufstatter I.U. Sauerheber R.D. Spragg R.G. Jackson J.H. Cochrane C.G. J. Biol. Chem. 1988; 263: 1665-1675Abstract Full Text PDF PubMed Google Scholar). As indicated above, eucaryotic gene expression may be controlled by the physical association of specific transcription factors with other proteins, such as PARP-1, which together form a multiprotein complex on enhancers and promoters (30Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar). For example, studies have recently suggested that PARP-1 participates in the regulation of eucaryotic transcription and gene expression, including NF-κB (17Meisterernst M. Stelzer G. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2261-2265Crossref PubMed Scopus (145) Google Scholar, 19Cervellera M.N. Sala A. J. Biol. Chem. 2000; 275: 10692-10696Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 31Le Page C. Sanceau J. Drapier J.C. Wietzerbin J. Biochem. Biophys. Res. Commun. 1998; 243: 451-457Crossref PubMed Scopus (106) Google Scholar, 32Hassa P.O. Hottiger M.O. Biol. Chem. 1999; 380: 953-959Crossref PubMed Scopus (265) Google Scholar, 33Oliver F.J. Menissier-de Murcia J. Nacci C. Decker P. Andriantsitohaina R. Muller S. de la Rubia G. Stoclet J.C. de Murcia G. EMBO J. 1999; 18: 4446-4454Crossref PubMed Scopus (548) Google Scholar). In fact, PARP-1 was shown to be required for proper NF-κB activation in lipopolysaccharide-treated mice (33Oliver F.J. Menissier-de Murcia J. Nacci C. Decker P. Andriantsitohaina R. Muller S. de la Rubia G. Stoclet J.C. de Murcia G. EMBO J. 1999; 18: 4446-4454Crossref PubMed Scopus (548) Google Scholar). Furthermore, the NF-κB activation-dependent transcription of nitric-oxide synthase was suppressed by PARP-1 inhibitors in murine macrophages (31Le Page C. Sanceau J. Drapier J.C. Wietzerbin J. Biochem. Biophys. Res. Commun. 1998; 243: 451-457Crossref PubMed Scopus (106) Google Scholar). However, the exact biochemical mechanism that mediated the PARP-1-dependent transcriptional activation was not shown. Therefore, to determine the biochemical role of PARP-1 in NF-κB activation in vitro, we co-incubated pure PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+. Our study illustrates the βNAD+-dependent binding of NF-κB-p50 to its oligodeoxynucleotide, a reduction in the DNA binding efficiency by 3-AB, and the physical interaction of PARP-1 with NF-κB-p50. Furthermore, our study also demonstrates a strong relationship between protein-poly(ADP-ribosyl)ation and NF-κB activation in oxidatively stressed HeLa cells. Construction of recombinant baculovirus containing cDNA of human PARP-1, its expression inSpodoptera frugiperda, and protein purification are described elsewhere (34Beneke S. Alvarez-Gonzalez R. Burkle A. Exp. Gerontol. 2000; 35: 989-1002Crossref PubMed Scopus (44) Google Scholar). For Fig. 4 B (see below), human PARP-1 and human NF-κB-p50 (Promega) were incubated for 20 min at room temperature in a mixture (20 μl) containing 100 mm Tris-HCl (pH 7.8), 10 mm MgCl2, 1 mm dithiothreitol, and 20 μg/ml synthetic octameric DNA (5′-GGAATTCC-3′, Integrated DNA Technologies) (35Grube K. Burkle A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11759-11763Crossref PubMed Scopus (264) Google Scholar). For this experiment, we used32P-labeled βNAD+ (ICN) as a substrate. The reaction was terminated by adding 2× SDS sample buffer, and proteins were fractionated through a 4–15% gradient polyacrylamide gel. Poly(ADP-ribosyl)ated proteins were visualized by autoradiography. For Fig. 4 A, pure PARP-1 and NF-κB-p50 were incubated for 20 min at room temperature in a mixture (20 μl) containing 20 mm Tris-HCl (pH 8.0), 60 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.05% Nonidet P-40, 10% glycerol, and 50 μg/ml bovine serum albumin (12Oei S.L. Griesenbeck J. Schweiger M. Ziegler M. J. Biol. Chem. 1998; 273: 31644-31647Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this case, we used 32P-labeled βNAD+as a substrate. Cold NF-κB oligodeoxynucleotide (≈2.5 ng of DNA) was then added, and the mixtures were further incubated for another 20 min. After terminating the reaction with 2× SDS sample buffer, proteins were fractionated by SDS-PAGE through a 4 to 15% gradient gel. For the radiolabeled probe of NF-κB, a duplex oligodeoxynucleotide containing the consensus sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′, Santa Cruz Biotechnology) was end-labeled with [γ-32P]ATP (ICN) and T4 DNA polynucleotide kinase (United States Biochemical Corp.). For EMSA with purified proteins, pure PARP-1 and NF-κB-p50 were incubated for 20 min at room temperature in a binding buffer containing 20 mm Tris-HCl (pH 8.0), 60 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.05% Nonidet P-40, 10% glycerol, and 50 μg/ml bovine serum albumin (12Oei S.L. Griesenbeck J. Schweiger M. Ziegler M. J. Biol. Chem. 1998; 273: 31644-31647Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In some reactions, βNAD+ (Roche Molecular Biochemicals), 3aminobenzamide (3-AB) (Sigma Chemical Co.), or duplex octameric DNA (5′-GGAATTCC-3′) was included. Equal amounts of32P-labeled NF-κB oligodeoxynucleotide (≈2.5 ng) were added, and the mixtures (20 μl) were incubated for another 20 min. Samples were separated at room temperature through a native 5% polyacrylamide gel containing 17.8 mm Tris borate and 0.4 mm EDTA. Protein-oligodeoxynucleotide complexes were visualized by autoradiography. For EMSA with HeLa nuclear extracts, cell treatment and nuclear extract preparation were done immediately before EMSA. Extracts containing 10 μg of protein each, 1 μg of poly(dI-dC), and 0.5 mm phenylmethylsulfonyl fluoride (PMSF) were incubated at room temperature for 10 min in the binding buffer before 32P-labeled NF-κB oligodeoxynucleotide (≈1.5 ng) were added. Reactions (20 μl) were incubated for another 15 min, and samples were electrophoresed through a native 5% polyacrylamide gel containing 45 mm Tris borate and 1 mm EDTA. For the identification of protein·oligodeoxynucleotide complex, 1 μg each of NF-κB-p50 antibody (Santa Cruz Biotechnology) or control antibody (PAb421 for p53; Oncogene Research) was incubated with nuclear extracts for 30 min at 4 °C before poly(dI-dC) and 32P-labeled NF-κB oligodeoxynucleotide were added. In the competition experiment shown in Fig. 1 below, we determined the off-rate for NF-κB-p50 from its DNA probe by EMSA also. To accomplish this, we incubated 80 ng each of NF-κB-p50 with pure32P-labeled NF-κB oligodeoxynucleotide (Promega) in the presence of increasing amounts of unlabeled oligodeoxynucleotide probe for 20 min at room temperature. Equimolar amounts of PARP-1 (800 ng) and NF-κB-p50 (350 ng) were incubated for 30 min at 4 °C in a buffer containing 10 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 0.1% Nonidet P-40 (19Cervellera M.N. Sala A. J. Biol. Chem. 2000; 275: 10692-10696Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). As a control experiment, NF-κB-p50 (350 ng) alone was also incubated. Protein was immunoprecipitated for 1 h at 4 °C with goat polyclonal antibody (Santa Cruz Biotechnology). The immune complexes were pulled-down by adding 30 μl of protein G-agarose beads (1:1 slurry) and incubating for 45 min at 4 °C with rocking. Beads were washed five times with the same buffer and adding 2× SDS sample buffer and boiling for 5 min eluted the bead-bound proteins. Proteins were fractionated by SDS-PAGE using an 8% gel under non-reducing conditions, and proteins were electrotransferred to a polyvinylidene difluoride membrane. The membrane was immunoblotted with rabbit anti-NF-κB-p50 polyclonal antibody (Santa Cruz Biotechnology) and NF-κB-p50 was detected with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Sigma) and an ECL chemiluminescence kit (Amersham Pharmacia Biotech). Human cervical adenocarcinoma cell line HeLa (CCL-2; American Type Culture Collection) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) at 37 °C in humidified 5% CO2 and air. For H2O2 and 3-AB treatment, exponentially growing cells were seeded in disc plates (≈0.4 × 106 cells/60-mm-diameter disc) 20–24 h before treatment. Cells were pre-treated with 10 mm 3-AB or its vehicle control for 1 h before treatment with H2O2for 1 h. H2O2 was diluted from 30% stock (Sigma) immediately before use. The vehicle did not interfere with NF-κB DNA binding (data not shown). Extracts were prepared by a modified method from Dignam et al. (36Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9168) Google Scholar) immediately before EMSA. Treated cells were washed with phosphate-buffered saline, harvested to microcentrifuge tubes, and briefly centrifuged (16,000 × g, 4 °C, 15 s). Cells were washed again with ice-cold phosphate-buffered saline, pelleted, and resuspended at 4 °C in a buffer containing 10 mm HEPES (pH 7.9), 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm PMSF, and protease inhibitors (5 μg each of aprotinin, leupeptin, and pepstatin per ml). Cells were allowed to swell on ice for 15 min, then Nonidet P-40 (0.15% final conc.) was added, and each sample was vigorously mixed. Nuclei were pelleted (16,000 ×g, 4 °C, 30 s) and resuspended in a buffer containing 20 mm HEPES (pH 7.9), 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol, 1 mm PMSF, and protease inhibitors (5 μg each of aprotinin, leupeptin, and pepstatin per milliliter). Nuclear lysates were maintained on ice for 15 min with occasional mixing. Nuclear extracts were cleared (16,000 × g, 4 °C, and 5 min) and transferred to new tubes. For 3-AB-pretreated cells, 10 mm of the inhibitor was included throughout the preparation of nuclear extracts. Protein concentrations were determined by Bradford assay. To be able to determine the molecular role of protein-poly(ADP-ribosyl)ation in the sequence specific binding of NF-κB-p50 binding to its consensus DNA sequence, we first proceeded to show the specificity of DNA binding of this polypeptide to its32P-radiolabeled DNA probe by electrophoretic mobility shift assays (EMSA). Fig. 1 A shows the off-rate of NF-κB-p50 from its radiolabeled probe following the addition of increasing amounts of unlabeled DNA probe to a fixed amount of its 32P-labeled oligodeoxynucleotide consensus sequence, and 80 ng of the DNA binding protein. After 20 min of incubation, the samples were fractionated through a 5% native polyacrylamide gel containing 0.2× Tris borate EDTA buffer and the protein·oligodeoxynucleotide complexes were visualized by autoradiography. As Fig. 1 A (lanes 1–8) shows, the mobility shift of the radiolabeled probe disappeared as a function of the amount of unlabeled oligodeoxynucleotide added. Therefore, from this experiment we conclude that our mobility shift test can be applied to study the role of protein-poly(ADP-ribosyl)ation in the sequence-specific DNA binding of NF-κB-p50. Fig. 1 B shows the disappearance of the mobility shift as a function of the concentration of unlabeled DNA added to the EMSA mixture and densitometric analysis of the data illustrated in Fig. 1 A. From the graphical representation observed here, it was clear that 50% of DNA binding specificity was lost when less than 1 pmol of unlabeled DNA probe was added, even after 20 min of incubation. These results are consistent with a strong and highly specific binding of pure NF-κB-p50 to the radiolabeled DNA probe used in these studies (see above under “Experimental Procedures”). Previous studies have suggested that PARP-1 may participate in NF-κB activation in various cell lines (31Le Page C. Sanceau J. Drapier J.C. Wietzerbin J. Biochem. Biophys. Res. Commun. 1998; 243: 451-457Crossref PubMed Scopus (106) Google Scholar, 32Hassa P.O. Hottiger M.O. Biol. Chem. 1999; 380: 953-959Crossref PubMed Scopus (265) Google Scholar, 33Oliver F.J. Menissier-de Murcia J. Nacci C. Decker P. Andriantsitohaina R. Muller S. de la Rubia G. Stoclet J.C. de Murcia G. EMBO J. 1999; 18: 4446-4454Crossref PubMed Scopus (548) Google Scholar, 37Kameoka M. Ota K. Tetsuka T. Tanaka Y. Itaya A. Okamoto T. Yoshihara K. Biochem. J. 2000; 346: 641-649Crossref PubMed Scopus (106) Google Scholar). To examine the effect of protein-poly(ADP-ribosyl)ation on NF-κB-p50 DNA binding in vitro, we co-incubated pure PARP-1 and NF-κB-p50 either in the presence or absence of βNAD+. The enzymatic activity of PARP-1 was allowed to proceed for 20 min at room temperature, and the 32P-labeled oligodeoxynucleotide containing the consensus sequence for NF-κB was added. To avoid a potential effect of exogenous DNA on NF-κB-p50 DNA binding, nicked DNA was omitted as an enzymatic activator of PARP-1. The binding of NF-κB-p50 to DNA was analyzed by subjecting the incubation reaction mixture to electrophoretic mobility shift assay (EMSA). Autoradiographic analysis showed the absence of mobility shift in the PARP-1 control (80 ng; Fig. 2, lane 1) in the presence of βNAD+. By contrast, we observed the presence of a single shift in the NF-κB-p50 control (80 ng; Fig. 2, lane 2), demonstrating the specificity of the NF-κB oligodeoxynucleotide. In the absence of βNAD+, adding PARP-1 (80 ng) to the NF-κB-p50 control caused an immediate reduction in the efficiency of binding of NF-κB-p50 to the oligodeoxynucleotide (Fig. 2, lane 3). When more PARP-1 (400 ng) was added to the mixture, a stronger inhibition of the specific DNA binding was observed, and this effect was accompanied by an apparent supershift of radiolabeled oligodeoxynucleotide (Fig. 2, lane 4). Whether this was an indication of competition between NF-κB-p50 and PARP-1 for the DNA probe or a reflection of protein·protein interactions was not initially clear. However, doubling the amount of PARP-1 to 800 ng (Fig. 2, lane 5) resulted in a stronger supershift and the complete absence of the typical mobility shift observed with NF-κB-p50 alone (compare with Fig. 2, lane 2). Interestingly, the PARP-1-dependent inhibition of NF-κB-p50 DNA binding was reversed by βNAD+ (200 μm) (Fig. 2, lanes 6–8). Indeed, the presence of βNAD+ abolished the inhibition of NF-κB-p50 binding to its oligodeoxynucleotide, presumably due to either the covalent poly(ADP-ribosyl)ation of NF-κB-p50, the automodification reaction of PARP-1, or both. Those NF-κB-p50·oligodeoxynucleotide complexes (Fig. 2, lanes 6–8) co-migrated with that of the NF-κB-p50 control (Fig. 2, lane 2), suggesting that NF-κB-p50 DNA binding became independent of PARP-1. Regardless of the PARP-1 concentration in the incubation mixture, the radiographic intensity of the NF-κB-p50·oligodeoxynucleotide complex (lanes 6–8) was similar to that of the NF-κB-p50 control (Fig. 2, lane 2). Therefore, these data suggest that there was no dilution effect of PARP-1 to NF-κB-p50 DNA binding. 3-Aminobenzamide (3-AB) is a well-established competitive inhibitor of βNAD+ in the protein-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1. Therefore, we next evaluated the efficiency of the βNAD+-dependent NF-κB-p50 DNA binding following co-incubation of PARP-1, NF-κB-p50, and βNAD+in the presence of 10 mm 3-AB (see above). Fig. 3shows that addition of 400 ng of PARP-1 inhibited NF-κB-p50 DNA binding in the absence of βNAD+. This inhibition was accompanied by an apparent supershift of radiolabeled oligodeoxynucleotide (Fig. 3, lane 3), as compared with NF-κB-p50 control (80 ng; Fig. 3, lane 2). As shown above, addition of 200 μm βNAD+ resulted in the nullification of the PARP-1 inhibitory effect and NF-κB-p50·oligodeoxynucleotide complex co-migrated with that of the NF-κB-p50 control (Fig. 3, lane 4). By contrast, inhibition of the auto-poly(ADP-ribosyl)ation reaction of PARP-1 with 10 mm 3-AB led to the characteristic inhibition of NF-κB-p50 DNA binding by native PARP-1 (Fig. 3, lane 5). These data demonstrates that the βNAD+-dependent NF-κB-p50 DNA binding is indeed the result of covalent protein-poly(ADP-ribosyl)ation. Overall, data shown in Figs. 2 and 3 indicate that PARP-1 interacts with NF-κB-p50 when PARP-1 is not poly(ADP-ribosyl)ated. Thus, when NF-κB-p50 interacts with PARP-1, it does not efficiently bind to its oligodeoxynucleotide. However, the auto-poly(ADP-ribosyl)ation of PARP-1 does not allow protein·protein interactions with NF-κB-p50, which in turn facilitates the DNA sequence-specific binding of the latter. We next proceeded to determine whether this effect observed on NF-κB-p50 DNA binding, as a result of the addition of PARP-1 and βNAD+ (Figs. 2 and 3), was due to the poly(ADP-ribosyl)ation of th