Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Functional Competition between Poly(ADP-ribose) Polymerase and Its 24-kDa Apoptotic Fragment in DNA Repair and Transcription

2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês

10.1074/jbc.m008044200

ISSN

1083-351X

Autores

Tetsu M.C. Yung, Masahiko S. Satoh,

Tópico(s)

Cell death mechanisms and regulation

Resumo

Poly(ADP-ribose) polymerase is a 113-kDa nuclear enzyme that binds to both damaged DNA and to RNA associated with actively transcribed regions of chromatin. Binding of poly(ADP-ribose) polymerase to DNA lesions activates it, catalyzing the covalent addition of multiple ADP-ribose polymers to the enzyme (automodification). During apoptosis, poly(ADP-ribose) polymerase is cleaved by caspase-3, resulting in the formation of an N-terminal 24-kDa fragment, containing the DNA binding domain, and a C-terminal 89-kDa catalytic fragment. The functional relevance of this cleavage is not well understood. We therefore prepared a recombinant 24-kDa poly(ADP-ribose) polymerase fragment and investigated the role of this fragment in DNA repair and transcription. The 24-kDa fragment retained its binding affinity for both DNA breaks and RNA. In an in vitro cell-free DNA repair assay, this fragment inhibited rejoining of DNA breaks and suppressed ADP-ribose polymer formation by competing with poly(ADP-ribose) polymerase in binding to DNA breaks. With regard to transcription, it has recently been demonstrated that binding of poly(ADP-ribose) polymerase to transcribed RNA reduces the rate of transcript elongation and that automodification of poly(ADP-ribose) polymerase bound to DNA breaks results in up-regulation of transcription. We tested the 24-kDa fragment for its ability to suppress transcript elongation, and we found that it competed against the up-regulation of transcription mediated by full-length poly(ADP-ribose) polymerase. The ability of the 24-kDa fragment to inhibit DNA repair, ADP-ribose polymer formation, and damage-dependent up-regulation of transcription may contribute to the apoptotic shift from cell survival to cell death mode. Poly(ADP-ribose) polymerase is a 113-kDa nuclear enzyme that binds to both damaged DNA and to RNA associated with actively transcribed regions of chromatin. Binding of poly(ADP-ribose) polymerase to DNA lesions activates it, catalyzing the covalent addition of multiple ADP-ribose polymers to the enzyme (automodification). During apoptosis, poly(ADP-ribose) polymerase is cleaved by caspase-3, resulting in the formation of an N-terminal 24-kDa fragment, containing the DNA binding domain, and a C-terminal 89-kDa catalytic fragment. The functional relevance of this cleavage is not well understood. We therefore prepared a recombinant 24-kDa poly(ADP-ribose) polymerase fragment and investigated the role of this fragment in DNA repair and transcription. The 24-kDa fragment retained its binding affinity for both DNA breaks and RNA. In an in vitro cell-free DNA repair assay, this fragment inhibited rejoining of DNA breaks and suppressed ADP-ribose polymer formation by competing with poly(ADP-ribose) polymerase in binding to DNA breaks. With regard to transcription, it has recently been demonstrated that binding of poly(ADP-ribose) polymerase to transcribed RNA reduces the rate of transcript elongation and that automodification of poly(ADP-ribose) polymerase bound to DNA breaks results in up-regulation of transcription. We tested the 24-kDa fragment for its ability to suppress transcript elongation, and we found that it competed against the up-regulation of transcription mediated by full-length poly(ADP-ribose) polymerase. The ability of the 24-kDa fragment to inhibit DNA repair, ADP-ribose polymer formation, and damage-dependent up-regulation of transcription may contribute to the apoptotic shift from cell survival to cell death mode. poly(ADP-ribose) polymerase N-methyl-N′-nitro-N-nitrosoguanidine enzyme-linked immunosorbent assay terminal dUTP nick-end labeling Dulbecco's modified Eagle's medium. Poly(ADP-ribose) polymerase (PARP)1 is a highly abundant nuclear enzyme present at about 2 × 105 molecules per nucleus (1Ludwig A. Behnke B. Holtlund J. Hilz H. J. Biol. Chem. 1988; 263: 6993-6999Abstract Full Text PDF PubMed Google Scholar). This enzyme is composed of an N-terminal DNA binding domain, containing two zinc finger motifs, a C-terminal NAD+ binding domain, catalyzing the synthesis of ADP-ribose polymers from its substrate, NAD+, and an automodification site, which unites the N-terminal and C-terminal domains (2de Murcia G. Ménissier-de Murcia J. Schreiber V. BioEssays. 1991; 13: 455-462Crossref PubMed Scopus (93) Google Scholar). Poly(ADP-ribosyl)ation by PARP at the automodification site of the protein is initiated by the binding of the zinc fingers to DNA breaks (3Althaus F.R. Richter C. ADP-ribosylation of Proteins: Enzymology and Biological Significance. Springer-Verlag, Berlin1987: 3-113Google Scholar, 4Lindahl T. Satoh M.S. Poirier G.G. Klungland A. Trends Biochem. Sci. 1995; 20: 405-411Abstract Full Text PDF PubMed Scopus (578) Google Scholar). As a consequence of this automodification, the binding affinity of PARP for DNA is reduced, resulting in dissociation of PARP from DNA breaks (5Zahradka P. Ebisuzaki K. Eur. J. Biochem. 1982; 127: 579-585Crossref PubMed Scopus (146) Google Scholar) and thereby allowing the DNA repair machinery to access the sites of DNA damage (6Satoh M.S. Lindahl T. Nature. 1992; 356: 356-358Crossref PubMed Scopus (975) Google Scholar). In cells where DNA breaks are generated by DNA-damaging agents, PARP is activated and automodified (3Althaus F.R. Richter C. ADP-ribosylation of Proteins: Enzymology and Biological Significance. Springer-Verlag, Berlin1987: 3-113Google Scholar, 4Lindahl T. Satoh M.S. Poirier G.G. Klungland A. Trends Biochem. Sci. 1995; 20: 405-411Abstract Full Text PDF PubMed Scopus (578) Google Scholar), leading to the conclusion that PARP is involved in the cellular response to genetic damage, particularly in the repair of damaged DNA (3Althaus F.R. Richter C. ADP-ribosylation of Proteins: Enzymology and Biological Significance. Springer-Verlag, Berlin1987: 3-113Google Scholar). However, PARP has been shown to lack DNA repair activity in itself (6Satoh M.S. Lindahl T. Nature. 1992; 356: 356-358Crossref PubMed Scopus (975) Google Scholar, 7Vodenicharov M.D. Sallmann F.R. Wang Z.-Q. Satoh M.S. Poirier G.G. Nucleic Acids Res. 2000; 28: 3887-3896Crossref PubMed Scopus (117) Google Scholar). Alternatively, it has been suggested that PARP is involved in chromatin stabilization (8Simbulan-Rosenthal C.M. Haddad B.R. Rosenthal D.S. Weaver Z. Coleman A. Luo R. Young H.M. Wang Z.Q. Ried T. Smulson M.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13191-13196Crossref PubMed Scopus (110) Google Scholar), in DNA replication (9Dantzer F. Nasheuer H.P. Vonesch J.L. de Murcia G. Menissier-de Murcia J. Nucleic Acids Res. 1998; 26: 1891-1898Crossref PubMed Scopus (138) Google Scholar, 10Simbulan-Rosenthal C.M. Rosenthal D.S. Boulares A.H. Hickey R.J. Malkas L.H. Coll J.M. Smulson M.E. Biochemistry. 1998; 37: 9363-9370Crossref PubMed Scopus (77) Google Scholar), and in transcription (11Kannan P., Yu, Y. Wankhade S. Tainsky M.A. Nucleic Acids Res. 1999; 27: 866-874Crossref PubMed Scopus (124) Google Scholar, 12Meisterernst M. Stelzer G. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2261-2265Crossref PubMed Scopus (145) Google Scholar, 13Anderson M.G. Scoggin K.E. Simbulan-Rosenthal C.M. Steadman J.A. J. Virol. 2000; 74: 2169-2177Crossref PubMed Scopus (64) Google Scholar), although the roles played by PARP in these processes are not yet understood. In nuclear localization experiments, PARP is observed in clear foci, associated both with regions of chromatin actively transcribed by RNA polymerase II as well as with nucleoli where rRNA is synthesized by RNA polymerase I (14Fakan S. Leduc Y. Lamarre D. Brunet G. Poirier G.G. Exp. Cell Res. 1988; 179: 517-526Crossref PubMed Scopus (31) Google Scholar). Dispersal of the foci upon treatment of cells with the transcription inhibitors actinomycin D or 5,6-dichloro-1-β-ribofuranosylbenzimidazole suggests an involvement of PARP in transcription (15Desnoyers S. Kaufmann S.H. Poirier G.G. Exp. Cell Res. 1996; 227: 146-153Crossref PubMed Scopus (58) Google Scholar). In addition, such foci are also dispersed by treatment of isolated nuclei with RNase (16Kaufmann S.H. Brunet G. Talbot B. Lamarr D. Dumas C. Shaper J.H. Poirier G. Exp. Cell Res. 1991; 192: 524-535Crossref PubMed Scopus (52) Google Scholar). These observations thus suggest an interaction between PARP and transcribed RNA. Recently, we demonstrated that RNA-bound PARP reduces the rate of RNA elongation by RNA polymerase II and that automodification of PARP in response to DNA damage up-regulates transcription (17Vispé S. Yung T.M.C. Ritchot J. Serizawa H. Satoh M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9886-9891Crossref PubMed Scopus (75) Google Scholar). Since DNA-damaging agents induce RNA damage as well, we proposed that this up-regulation allows cells to compensate for the loss of damaged RNA that occurs collaterally with DNA damage and that this pathway is required for cell survival following exposure to DNA-damaging agents (17Vispé S. Yung T.M.C. Ritchot J. Serizawa H. Satoh M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9886-9891Crossref PubMed Scopus (75) Google Scholar). When cells are exposed to sufficiently high levels of DNA-damaging agents, they commit to cell death by inducing either apoptosis or necrosis. During apoptosis PARP is cleaved by the apoptosis-specific protease, caspase-3, resulting in the formation of an N-terminal 24-kDa fragment, containing the DNA binding domain, and a C-terminal 89-kDa catalytic domain, containing the automodification site (18Kaufmann S.H. Cancer Res. 1989; 49: 5870-5878PubMed Google Scholar, 19Duriez P.J. Shah G.M. Biochem. Cell Biol. 1997; 75: 337-349Crossref PubMed Scopus (417) Google Scholar, 20Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2351) Google Scholar). Recently, Halappanavar et al. (21Halappanavar S.S. Rhun Y.L. Mounir S. Martins L.M. Huot J. Earnshaw W.C. Shah G.M. J. Biol. Chem. 1999; 274: 37097-37104Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and Oliver et al. (22Oliver F.J. de la Rubia G. Rolli V. Ruiz-Ruiz M.C. de Murcia G. Murcia J.M. J. Biol. Chem. 1998; 273: 33533-33539Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar) reported that, in PARP knockout cells, expression of uncleavable PARP, lacking the caspase-3 recognition sequence, causes delayed induction of DNA damage-induced apoptosis. This observation suggests that cleavage of PARP has a role in damage-induced apoptosis. In addition, Herceg and Wang (23Herceg Z. Wang Z.Q. Mol. Cell. Biol. 1999; 19: 5124-5133Crossref PubMed Scopus (223) Google Scholar) and Boulares et al. (24Boulares A.H. Yakovlev A.G. Ivanova V. Stoica B.A. Wang G. Iyer S. Smulson M. J. Biol. Chem. 1999; 274: 22932-22940Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar) suggested that cleavage of PARP is also required for tumor necrosis factor-α-induced cell death. However Herceg and Wang (23Herceg Z. Wang Z.Q. Mol. Cell. Biol. 1999; 19: 5124-5133Crossref PubMed Scopus (223) Google Scholar) found increased cell death by necrosis, whereas Boulares et al.(24Boulares A.H. Yakovlev A.G. Ivanova V. Stoica B.A. Wang G. Iyer S. Smulson M. J. Biol. Chem. 1999; 274: 22932-22940Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar) demonstrated a promotion of apoptotic, rather than necrotic, cell death by expressing uncleavable PARP in PARP knockout cells. Since the 24-kDa fragment contains the DNA binding domain, which is capable of binding to DNA breaks (25Smulson M.E. Pang D. Jung M. Dimtchev A. Chasovskikh S. Spoonde A. Simbulan-Rosenthal C. Rosenthal D. Yakovlev A. Dritschilo A. Cancer Res. 1998; 58: 3495-3498PubMed Google Scholar), it has been speculated that the 24-kDa fragment possibly counteracts functions of PARP and promotes the process of apoptosis (19Duriez P.J. Shah G.M. Biochem. Cell Biol. 1997; 75: 337-349Crossref PubMed Scopus (417) Google Scholar). However, biochemical characteristics of the 24-kDa fragment remain to be elucidated. Thus, we prepared the 24-kDa apoptotic fragment of PARP and asked whether the 24-kDa fragment competes against the functions of PARP in DNA repair, ADP-ribose polymer formation, and transcription. GMO1953A lymphoblastoid cells were obtained from NIGMS Human Mutant Cell Repository (Camden, NJ). The C-II-10 antibody against the automodification domain of PARP and the F1–23 antibody against the DNA binding domain of PARP (zinc finger 2) (26Duriez P.J. Desnoyers S. Hoflack J.C. Shah G.M. Morelle B. Bourassa S. Poirier G.G. Talbot B. Biochim. Biophys. Acta. 1997; 1334: 65-72Crossref PubMed Scopus (48) Google Scholar) were kindly provided by Dr. G. G. Poirier. Full-length PARP cDNA (bases 1–3039) or the sequence corresponding to the 24-kDa DNA binding domain of PARP (bases 1–654), which is found in apoptotic cells (18Kaufmann S.H. Cancer Res. 1989; 49: 5870-5878PubMed Google Scholar, 19Duriez P.J. Shah G.M. Biochem. Cell Biol. 1997; 75: 337-349Crossref PubMed Scopus (417) Google Scholar, 20Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2351) Google Scholar), was cloned into pET3a (Novagen) and used to transform HMS 174 de3 cells (Novagen) together with pLysE (Novagen). After overnight pre-culture, theEscherichia coli were propagated in 2 liters of Luria-Bertani medium in the presence of 34 μg/ml chloramphenicol and 100 μg/ml ampicillin for 3 h, and expression of PARP or the 24-kDa fragment was induced in the presence of 0.4 mmisopropyl-β-d-thiogalactoside for 3 h at 37 °C. The bacteria were then spun down at 3500 × g for 10 min, and the pellet was washed in phosphate-buffered saline and spun down. The resulting pellet was resuspended in 20 ml of buffer containing 0.1 m NaCl, 50 mm Tris-HCl, pH 8.0, 12% glycerol, 2 mm MgCl2, and 0.1 mm phenylmethylsulfonyl fluoride (Buffer CB), and PARP or the 24-kDa fragment was extracted by sonication. After a 30-min centrifugation at 35,000 × g (at 4 °C), the supernatant was used for purification of PARP or the 24-kDa fragment. E. coli lysate (750 mg) was applied to a phosphocellulose column (10 mm diameter and 2-ml bed volume) equilibrated with Buffer CB. PARP was eluted using a linear gradient of Buffer CB containing 0.1–2.0 m NaCl. The fractions of interest were determined by silver stain analysis of samples run on SDS-12.5% polyacrylamide gels. The fractions of interest were then pooled, diluted with Buffer CB to reduce the NaCl concentration below 0.2 m, and applied to a DNA cellulose column (5 mm diameter and 2-ml bed volume) equilibrated with Buffer CB. After washing the column with 20 ml of Buffer CB followed by 8 ml of Buffer CB containing 0.4 m NaCl, PARP was eluted with 8 ml of Buffer CB containing 1.0 m NaCl. The eluate was diluted with Buffer CB to reduce the NaCl concentration below 0.2 mand applied to a heparin-Sepharose column (Amersham Pharmacia Biotech, Hi-Trap, 5 ml) equilibrated with Buffer CB. PARP was eluted using a linear gradient of Buffer CB containing 0.1–2.0 m NaCl, and the final fractions were dialyzed against Buffer CB prior to analysis. Purification was carried out as for PARP except that the DNA cellulose chromatography was omitted. The final preparation was dialyzed against Buffer CB prior to analysis. The purified recombinant PARP and the 24-kDa fragment were quantified by direct ELISA. Briefly, PARP and the 24-kDa fragment were immobilized on a microtiter plate, treated with the antibody F1–23 (26Duriez P.J. Desnoyers S. Hoflack J.C. Shah G.M. Morelle B. Bourassa S. Poirier G.G. Talbot B. Biochim. Biophys. Acta. 1997; 1334: 65-72Crossref PubMed Scopus (48) Google Scholar) (5000-fold dilution) which recognizes the zinc finger 2 motif present in both PARP and the 24-kDa fragment, and then treated with secondary antibody conjugated to horseradish peroxidase. Following addition of TMB substrate (Research Diagnostics Inc.), 0.18m H2SO4 was used to initiate the peroxidase reaction. PARP and the 24-kDa fragment were quantified based on absorbance at 490 nm. To prepare the DNA probe for the gel retardation assay, a single-stranded 50-base oligodeoxynucleotide (100 pmol) was 32P-labeled at 37 °C for 15 min using 25 μCi of [γ-32P]ATP with 10 units of T4 polynucleotide kinase (Amersham Pharmacia Biotech) in a 25-μl reaction mixture using the buffer provided by the supplier. The labeled single-stranded oligodeoxynucleotide was annealed with its complementary single-stranded oligodeoxynucleotide (50 bases) in a 100-μl mixture containing 10 mm Tris-HCl, pH 8.0, and 1 mm EDTA by incubating for 5 min at 95 °C, followed by 5 min at 50 °C, and then 20 min at 22 °C. The annealed double-stranded oligodeoxynucleotide was then precipitated with ethanol and ammonium acetate, and the resulting pellet was dissolved in 10 mm Tris-HCl, pH 8.0, and 1 mm EDTA. The binding reaction was carried out using 1 pmol of the 32P-labeled double-stranded oligodeoxynucleotide with varying amounts of PARP or the 24-kDa fragment in a buffer containing 5 mm Tris-HCl, pH 8.0, and 5 mm MgCl2 for 15 min at 30 °C in a 15-μl reaction mixture. Samples were then fractionated by native 6% polyacrylamide gel electrophoresis, and the gel was dried and exposed to x-ray film for autoradiography or used for quantitation by AlphaImager (Packard Instrument Co.). Gel retardation assays using an RNA probe were carried out as described previously (17Vispé S. Yung T.M.C. Ritchot J. Serizawa H. Satoh M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9886-9891Crossref PubMed Scopus (75) Google Scholar). Briefly,32P-labeled stem-loop RNA was synthesized using T3 RNA polymerase to transcribe the human immunodeficiency virus, type I TAR sequence. The 32P-labeled stem-loop RNA was then incubated with PARP or the 24-kDa fragment and fractionated by native 6% polyacrylamide gel electrophoresis. After drying, the gel was either exposed to x-ray film for autoradiography or used for quantitation by AlphaImager (Packard Instrument Co.). Cell-free extracts were prepared from GMO1953A lymphoblastoid cells following the method of Manleyet al. (27Manley J.L. Fire A. Samuels M. Sharp P.A. Methods Enzymol. 1983; 101: 568-582Crossref PubMed Scopus (222) Google Scholar). The cell-free DNA repair assay was carried out using 50 μg of extract, 300 ng of γ-irradiated pBluescript II KS+ (pBS, 3 kilobase pairs) containing an average of one single-stranded DNA break per molecule (6Satoh M.S. Lindahl T. Nature. 1992; 356: 356-358Crossref PubMed Scopus (975) Google Scholar), and varying amounts of the 24-kDa fragment in the presence or absence of 2 mmNAD+ under reaction conditions described previously (28Satoh M.S. Poirier G.G. Lindahl T. J. Biol. Chem. 1993; 268: 5480-5487Abstract Full Text PDF PubMed Google Scholar). After purification of the DNA, unrepaired pBS (open circular) and repaired pBS (closed circular) were resolved by ethidium bromide, 1% agarose gel electrophoresis. To determine the amount of ADP-ribose polymers produced in the cell-free DNA repair assay, 1.3 μCi of [32P]NAD+ and 0.25 mmNAD+ were added to the reaction described above. Reactions were terminated by addition of trichloroacetic acid, and insoluble32P activity retained on a GF/C filter was counted as described previously (28Satoh M.S. Poirier G.G. Lindahl T. J. Biol. Chem. 1993; 268: 5480-5487Abstract Full Text PDF PubMed Google Scholar). Pulse-chase elongation assays were carried out as described previously (17Vispé S. Yung T.M.C. Ritchot J. Serizawa H. Satoh M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9886-9891Crossref PubMed Scopus (75) Google Scholar). Briefly, a pGf1 plasmid containing a G-less sequence was digested with PacI, creating a 90-base G-less sequence at one end of the DNA. A C-tail was added to allow loading of RNA polymerase II from DNA break ends. The DNA was then incubated with RNA polymerase II (1.0 units) (provided by Dr. H. Serizawa) (29Serizawa H. Makela T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Yung R.A. Nature. 1995; 374: 280-282Crossref PubMed Scopus (309) Google Scholar) in the presence of RNasin, ATP, UTP, CTP, and [α-32P]CTP at 37 °C for 30 min as described by Shilatifard et al. (30Shilatifard A. Lane W.S. Jackson K.W. Conaway R.C. Conaway J.W. Science. 1996; 271: 1873-1876Crossref PubMed Scopus (281) Google Scholar). A chase was initiated by addition of GTP and excess CTP in the presence or absence of PARP or the 24-kDa fragment. After extraction and precipitation, RNA was fractionated on a 6% polyacrylamide, 8 m urea gel. After drying, the gel was exposed to x-ray film for autoradiography. pCMV-Luc containing the cytomegalovirus promoter was linearized by ScaI restriction digestion and used in a run-off transcription assay with HeLa nuclear extracts (Promega). The expected size of the run-off product was 400 bases. Reactions were carried out for 1 h at 30 °C using 4 μg/ml linearized pCMV-Luc in a reaction mixture containing 10 mm HEPES-KOH, pH 7.9, 40 mm KCl, 0.2 mm dithiothreitol, 3 mm MgCl2, 400 μCi/ml [α-32P]GTP, 400 μm ATP, 400 μm CTP, 400 μm UTP, 16 μmGTP, 10% glycerol, 1000 transcription units/ml of HeLa nuclear extract, and varying amounts of either PARP or the 24-kDa fragment. The reaction was terminated by addition of 7 volumes of 0.3 mTris-HCl, pH 7.4, 0.3 m sodium acetate, 0.5% SDS, 2 mm EDTA, and 3 μg/ml tRNA. Following phenol/chloroform extraction, transcripts were precipitated with ethanol, fractionated by 3% polyacrylamide, 8 m urea gel electrophoresis, and visualized by autoradiography. The mammalian expression vector pcDNA 3.1− (Invitrogen) was used to clone the cDNA corresponding to the 24-kDa DNA binding domain of PARP (pcDNA 3.1−/AF24). Then 1.25 μg/ml pcDNA3.1-/AF24 was incubated with 3.75 μl/ml Tfx20™ reagent (Promega) in serum-free Dulbecco's modified Eagle's medium (serum-free DMEM) for 10 min at room temperature and used to treat HeLa S3 cells (attached to coverslips) for 1 h at 37 °C. Following cell treatment, DMEM containing 10% fetal bovine serum and antibiotics (complete DMEM) was added; HeLa S3 cells were cultured for 24 h after which the medium was replaced with serum-free DMEM, and the cells were exposed toN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (50 μm). After 20 min of treatment at 37 °C, the medium was replaced with complete DMEM, and cells were cultured for 2 h. TUNEL staining (fluorescein in situ cell death detection kit, Roche Molecular Biochemicals) was then carried out after fixing the cells in 3% paraformaldehyde and permeabilizing them with 0.1% Triton X-100 and 0.1% sodium citrate according to the supplier's instructions (Roche Molecular Biochemicals). TUNEL-positive cells were visualized by fluorescence microscopy. To investigate the effect of the 24-kDa PARP fragment on DNA repair and transcription, we first prepared the recombinant 24-kDa fragment and full-length PARP. As shown in Fig. 1, the 24-kDa fragment, which migrated to an apparent molecular mass of about 30 kDa on SDS-polyacrylamide gels, was purified to over 99% homogeneity as described under "Materials and Methods". The purity of full-length recombinant PARP was about 95%, with several truncated products observed (Fig. 1). Quantitation of the 24-kDa fragment and PARP was carried out by direct ELISA using the F1–23 antibody (26Duriez P.J. Desnoyers S. Hoflack J.C. Shah G.M. Morelle B. Bourassa S. Poirier G.G. Talbot B. Biochim. Biophys. Acta. 1997; 1334: 65-72Crossref PubMed Scopus (48) Google Scholar), which recognizes the zinc finger 2 motif of PARP and the 24-kDa fragment. 32P-Labeled double-stranded oligodeoxynucleotide (50 base pairs) was incubated with either the 24-kDa fragment or PARP. If the zinc finger motifs found in PARP and the 24-kDa fragment bind to DNA ends (2de Murcia G. Ménissier-de Murcia J. Schreiber V. BioEssays. 1991; 13: 455-462Crossref PubMed Scopus (93) Google Scholar, 31D'Silva I. Pelletier J.D. Lagueux J. D'Amours D. Chaudhry M.A. Weinfeld M. Lees-Miller S.P. Poirier G.G. Biochim. Biophys. Acta. 1999; 1430: 119-126Crossref PubMed Scopus (89) Google Scholar), the mobility of the32P-labeled DNA probe should be reduced on a native gel. As shown in Fig. 2, discrete retarded bands were in fact observed when the labeled probe was incubated with the 24-kDa fragment. Migration of the probe was similarly retarded when PARP was used instead of the 24-kDa fragment, with the probe migrating slightly more slowly than in the case of the 24-kDa fragment. In addition, the labeled probe was reproducibly found at the origin of the lane. Addition of excess unlabeled double-stranded oligodeoxynucleotide inhibited retardation of the probe, confirming that formation of the retarded labeled material was due to binding of the 24-kDa fragment or PARP to the DNA probe (data not shown). The data in Fig. 2 reveal a linear relationship between 32P activity associated with the retarded fractions and the amount of the 24-kDa fragment or PARP and allow us to determine that the 24-kDa fragment has about 25% of the binding activity of full-length PARP. Thus, even after cleavage of PARP by caspase-3, the resulting 24-kDa fragment retains significant DNA binding activity. In the absence of PARP's substrate, NAD+, PARP binds to and persists on DNA breaks, thereby inhibiting DNA repair (6Satoh M.S. Lindahl T. Nature. 1992; 356: 356-358Crossref PubMed Scopus (975) Google Scholar). Thus, dissociation of PARP from DNA breaks by automodification is a prerequisite for DNA repair (6Satoh M.S. Lindahl T. Nature. 1992; 356: 356-358Crossref PubMed Scopus (975) Google Scholar). Since the 24-kDa fragment is capable of binding to DNA breaks (Fig. 2) but lacks the automodification site, these fragments should persist on DNA breaks and inhibit DNA repair even in the presence of NAD+. To test this hypothesis, a cell-free DNA repair assay was carried out using open circular pBS containing γ-ray-induced single-stranded DNA breaks, cell-free extracts, and varying amounts of the 24-kDa fragment in the presence or absence of NAD+. As shown in Fig.3, only 7% of DNA breaks were rejoined in the absence of NAD+ due to inhibition of DNA repair by bound PARP. By contrast, when poly(ADP-ribosyl)ation and dissociation of PARP from DNA breaks was initiated by addition of NAD+, about 30% of DNA breaks were repaired. This NAD+-promoted DNA repair was significantly inhibited by addition of the 24-kDa fragment (Fig. 3, A and B). A 6.6-fold molar excess of the 24-kDa fragment relative to PARP was sufficient to inhibit NAD+-promoted DNA repair by 80% (Fig. 3,A and B, 15 pmol of PARP derived from extractversus 100 pmol of the 24-kDa fragment), consistent with the hypothesis that the 24-kDa fragment, unlike full-length PARP, binds to and persists on DNA breaks in the presence of NAD+. We then measured the amount of ADP-ribose polymers generated in the cell-free assay in the absence or presence of the recombinant 24-kDa fragment. As previously observed (28Satoh M.S. Poirier G.G. Lindahl T. J. Biol. Chem. 1993; 268: 5480-5487Abstract Full Text PDF PubMed Google Scholar), incubation of cell-free extracts with DNA breaks caused transient formation of ADP-ribose polymers (Fig.3 C). However, addition of the 24-kDa fragment significantly inhibited ADP-ribose polymer formation, suggesting that the 24-kDa fragment effectively competed with PARP in binding to DNA breaks and thereby reduced the overall level of poly(ADP-ribosyl)ation in the presence of NAD+. Since ADP-ribose polymers are synthesized from NAD+, inhibition of poly(ADP-ribose) formation should reduce the consumption of NAD+. To quantify the amount of NAD+ present in the reaction mixture following the cell-free DNA repair assay, [32P]NAD+ (32 nm) was added to the assay and the reaction mixtures were applied to a 20% polyacrylamide, 8 m urea gel (32Satoh M.S. Hanawalt P.C. Nucleic Acids Res. 1996; 24: 3576-3582Crossref PubMed Scopus (17) Google Scholar) for quantification of labeled NAD+. Activation of PARP consumed 20% of the available NAD+, whereas addition of the 24-kDa fragment (200 pmol) resulted in only a negligible reduction in NAD+levels (data not shown). Taken together, these results suggest that the 24-kDa fragment inhibits DNA repair and ADP-ribose formation by binding to and persisting on DNA breaks in competition with full-length PARP. In nuclei, PARP has been observed to associate with chromatin, particularly with actively transcribed regions; this association has been shown to occur by direct binding of PARP to transcribed RNA (14Fakan S. Leduc Y. Lamarre D. Brunet G. Poirier G.G. Exp. Cell Res. 1988; 179: 517-526Crossref PubMed Scopus (31) Google Scholar, 15Desnoyers S. Kaufmann S.H. Poirier G.G. Exp. Cell Res. 1996; 227: 146-153Crossref PubMed Scopus (58) Google Scholar, 16Kaufmann S.H. Brunet G. Talbot B. Lamarr D. Dumas C. Shaper J.H. Poirier G. Exp. Cell Res. 1991; 192: 524-535Crossref PubMed Scopus (52) Google Scholar). We recently demonstrated that binding of PARP to RNA stem-loop structures reduces the rate of RNA elongation by RNA polymerase II and that formation of DNA breaks and subsequent automodification of PARP removes the transcriptionally inhibitory PARP molecules, thus up-regulating RNA synthesis (17Vispé S. Yung T.M.C. Ritchot J. Serizawa H. Satoh M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9886-9891Crossref PubMed Scopus (75) Google Scholar). To investigate whether the 24-kDa fragment binds to RNA stem-loops, uniformly 32P-labeled synthetic stem-loop RNA was prepared by transcribing the TAR sequence from human immunodeficiency virus, type I (see "Materials and Methods"), and was mixed with either the 24-kDa fragment or PARP. As shown in Fig.4, PARP reduced the mobility of stem-loop RNA on a native polyacrylamide gel, generating a discrete band. The 24-kDa fragment also reduced the mobility of TAR stem-loop RNA (Fig.4), suggesting that the fragment, like full-length PARP

Referência(s)