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• W2022388411 abstract "Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression. Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression. DNA topoisomerase I (topo I) 2The abbreviations used are: topo I, DNA topoisomerase I; ASF/SF2, alternative splicing factor/splicing factor 2; CPT, camptothecin; PAR, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; SRPK1, SR-protein specific kinase 1; GST, glutathione S-transferase; DTT, dithiothreitol; RRM, RNA recognition motif; ds, double strand. is a constitutively expressed multifunctional enzyme that localizes at active transcription sites (1Zhang H. Wang J.C. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1060-1064Crossref PubMed Scopus (262) Google Scholar, 2Khobta A. Ferri F. Lotito L. Montecucco A. Rossi R. Capranico G. J. Mol. Biol. 2006; 357: 127-138Crossref PubMed Scopus (49) Google Scholar). Its best known function is to control the topological state of DNA by relieving torsional stress that is generated following DNA strand separation during transcription, replication, and repair (3Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1901) Google Scholar, 4Leppard J.B. Champoux J.J. Chromosoma. 2005; 114: 75-85Crossref PubMed Scopus (179) Google Scholar, 5Pommier Y. Nat. Rev. Cancer. 2006; 6: 789-802Crossref PubMed Scopus (1628) Google Scholar). The catalytic mechanism involves the formation of a DNA·topo I complex (cleavage complex) with the enzyme being covalently bound to the 3′-end of the cleaved DNA strand through a tyrosine-phosphate ester bond. Cleavage complexes are usually short-lived; their stabilization by compounds of the camptothecin (CPT) family of anticancer drugs may cause DNA strand break accumulation and eventually lead to cell death. Human topo I can relax both negative and positive supercoils by controlled rotation of the DNA strand downstream of the cleavage site followed by break resealing and restoration of an intact DNA duplex. In addition to relaxing supercoiled DNA, human topo I also plays a major role in pre-mRNA splicing, being endowed with a protein kinase activity targeted at a group of splicing factors of the serine-arginine (SR)-rich protein family (6Rossi F. Labourier E. Forné T. Divita G. Derancourt J. Riou J.F. Antoine E. Cathala G. Brunel C. Tazi J. Nature. 1996; 381: 80-82Crossref PubMed Scopus (287) Google Scholar). SR-proteins function both as components of the basal RNA splicing machinery and as regulators of alternative splicing (7Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (880) Google Scholar, 8Sanford J.R. Ellis J. Caceres J.F. Biochem. Soc. Trans. 2005; 33: 443-446Crossref PubMed Scopus (126) Google Scholar). Moreover, SR-proteins are involved in the control of mRNA transport and stability (8Sanford J.R. Ellis J. Caceres J.F. Biochem. Soc. Trans. 2005; 33: 443-446Crossref PubMed Scopus (126) Google Scholar, 9Huang Y. Steitz J.A. Mol. Cell. 2005; 17: 613-615Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) and contribute to the maintenance of genomic stability (10Li X. Manley J.L. Cell. 2005; 122: 365-378Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). SR-proteins are structurally characterized by having one or two domains that include a RNA recognition motif (RRM) at the N terminus, and an SR-rich C-terminal domain (RS domain) containing a variable number of SR dipeptidic repeats. Phosphorylation at serine residues in such sequences regulates SR-protein functions as well as their subnuclear localization (7Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (880) Google Scholar, 8Sanford J.R. Ellis J. Caceres J.F. Biochem. Soc. Trans. 2005; 33: 443-446Crossref PubMed Scopus (126) Google Scholar, 9Huang Y. Steitz J.A. Mol. Cell. 2005; 17: 613-615Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 11Sanford J.R. Ellis J.D. Cazalla D. Caceres J.F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15042-15047Crossref PubMed Scopus (103) Google Scholar, 12Stamm S. J. Biol. Chem. 2008; 283: 1223-1227Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Besides topo I, other kinases are also involved in SR-protein phosphorylation; these include the SR-protein-specific kinases 1 and 2 (SRPK1 and SRPK2) and the cell cycle-dependent dual specificity kinase Clk/Sty (13Colwill K. Feng L.L. Yeakley J.M. Gish G.D. Caceres J.F. Pawson T. Fu X.-D. J. Biol. Chem. 1996; 271: 24569-24575Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Moreover, Akt/protein kinase B phosphorylation of SR-protein family members appears to play a critical role in signal transduction pathways linking extracellular stimuli (hormones and mitogens) to changes in gene expression via regulation of alternative splicing and mRNA translation (14Patel N.A. Kaneko S. Apostolatos H.S. Bae S.S. Watson J.E. Davidowitz K. Chappell D.S. Birnbaum M.J. Cheng J.Q. Cooper D.R. J. Biol. Chem. 2005; 280: 14302-14309Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 15Blaustein M. Pelish F. Tanos T. Munoz M.J. Wengier D. Quadrana L. Sanford J.R. Muschietti J.P. Kornblihtt A.R. Caceres J.F. Coso O.A. Srebrow A. Nat. Struct. Mol. Biol. 2005; 12: 1037-1044Crossref PubMed Scopus (190) Google Scholar). Depletion of topo I results in the hypophosphorylation of SR-proteins and impaired exonic enhancer-dependent splicing (16Soret J. Gabut M. Dupon C. Kohlhagen G. Stévenin J. Pommier Y. Tazi J. Cancer Res. 2003; 63: 8203-8211PubMed Google Scholar). Likewise, inhibition of topo I-dependent SR-protein phosphorylation by indolocarbazole antitumor drugs has been shown to interfere with the spliceosome assembly pathway, leading to altered gene expression and eventually to cell death (17Pilch B. Allemand E. Facompre M. Bailly C. Riou J.F. Soret J. Tazi J. Cancer Res. 2001; 61: 6876-6884PubMed Google Scholar). Thus, human topo I may either bind DNA and catalyze its relaxation or bind SR-proteins and ATP and play the role of a kinase. The two activities are mutually exclusive, and they are most likely the functional expression of distinct conformational states (18Chen H.J. Hwang J. Eur. J. Biochem. 1999; 265: 367-375Crossref PubMed Scopus (19) Google Scholar). What regulates such structural and functional transitions is unknown. Human topo I is a member of the poly-(ADP-ribose)-binding family of proteins (19Pleschke J. Kleczkowska H. Strohm M. Althaus F.R. J. Biol. Chem. 2000; 275: 40974-40980Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). Poly(ADP-ribose) (PAR) is the product of a class of enzymes known as PAR polymerases (PARPs) (20Amé J.C. Spenlehauer C. de Murcia G. BioEssays. 2004; 26: 882-893Crossref PubMed Scopus (1231) Google Scholar). PARPs utilize NAD+ as a source of ADP-ribose units and catalyze the covalent modification of a number of proteins (heteromodification), including themselves (automodification), with an array of linear or branched ADP-ribose chains of variable lengths; these polymers are then degraded by a specific PAR glycohydrolase, thus making the reaction reversible (20Amé J.C. Spenlehauer C. de Murcia G. BioEssays. 2004; 26: 882-893Crossref PubMed Scopus (1231) Google Scholar, 21Burkle A. FEBS J. 2005; 272: 4576-4589Crossref PubMed Scopus (249) Google Scholar). PARP-1 and PARP-2, the best known members of the PARP family, depend on DNA strand breaks for activity and are responsible for most PAR synthesized in the nucleus of eukaryotic cells both under physiological and DNA damage conditions (20Amé J.C. Spenlehauer C. de Murcia G. BioEssays. 2004; 26: 882-893Crossref PubMed Scopus (1231) Google Scholar, 21Burkle A. FEBS J. 2005; 272: 4576-4589Crossref PubMed Scopus (249) Google Scholar). Protein targeting by PARP-bound polymers via non-covalent, yet specific interactions, is emerging as an important regulatory mechanism for diverse biological functions, including transcription, DNA damage signaling and checkpoint activation, proteasomal histone degradation, and mitotic spindle formation (22Malanga M. Althaus F.R. Biochem. Cell Biol. 2005; 83: 354-364Crossref PubMed Scopus (226) Google Scholar, 23Schreiber V. Dantzer F. Amé J.C. de Murcia G. Nat. Rev. Mol. Cell Biol. 2006; 7: 517-528Crossref PubMed Scopus (1574) Google Scholar). topo I bears three PAR-binding sites localized in domains that are critical for the catalytic activity of the enzyme on DNA and for its regulation (24Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In fact, PAR has a dual effect on topo I: it inhibits DNA cleavage (thus preventing initiation of new catalytic cycles) while it stimulates the re-ligation activity of the enzyme blocked in a ternary complex with nicked DNA and CPT (thus counteracting the poisoning effect of the drug) (24Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In this study we addressed the question of whether PAR could also affect topo I kinase activity and/or act as a molecular switch of distinct topo I functions. ASF/SF2, a prototype of the SR-protein family, was used as a specific substrate for the topo I kinase activity. Purified human topo I was obtained from TopoGen. This enzyme undergoes spontaneous conversion into a 70-kDa form lacking the N-terminal domain (ΔN-topo I). Full-length His-tagged human topo I expressed in a baculovirus system was from Jena Bioscience (distributed by Alexis). topo I homogeneity was >95%. GST-SRPK1 was from Upstate. Highly purified recombinant PARP-1 and PARP-2 were purchased from Alexis. Protein-free, affinity-purified [14C]PAR, [32P]PAR, and 32P-5′-end-labeled ds-oligonucleotide, used as a topo I substrate in DNA cleavage assays, were prepared as previously described (24Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). All experiments were repeated at least three times and confirmed with different preparations of topo I, SR-proteins, PAR. Recombinant Proteins—Human topo I was expressed in Saccharomyces cerevisiae EKY3 strain and purified essentially as described by Kowalska-Loth et al. (25Kowalska-Loth B. Girstun A. Piekielko A. Staron K. Eur. J. Biochem. 2002; 269: 3504-3510Crossref PubMed Scopus (20) Google Scholar). Briefly, the expression of topo I was induced with 2% galactose. After 4 h, cells were harvested, washed once with SED buffer (1 m sorbitol, 25 mm EDTA, pH 8.0, 50 mm dithiothreitol (DTT), and treated with 5 mg/ml zymolyase 100T (Seikagaku) for 30 min at 30 °C. Spheroplasts were pelleted by centrifugation and extracted twice with YLS buffer (20 mm Tris, pH 7.5, 0.5 m KCl, 1 mm EDTA, 1 mm DTT, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride). Yeast extracts were adjusted to buffer B conditions (0.35 m NaCl, 25 mm Tris-HCl, pH 7.5, 3 mm MgCl2, 10 mm β-mercaptoethanol, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride), loaded onto a heparin-agarose column (Bio-Rad), and eluted by increasing salt concentration. topo I-containing fractions were pooled and loaded onto a nickel-nitrilotriacetic acid-agarose column (Qiagen) for further purification. Finally, purified topo I was concentrated on Biomax-5K columns (Millipore). His-tagged SF2 was expressed in Escherichia coli TG1 strain and purified as described by Rossi et al. (6Rossi F. Labourier E. Forné T. Divita G. Derancourt J. Riou J.F. Antoine E. Cathala G. Brunel C. Tazi J. Nature. 1996; 381: 80-82Crossref PubMed Scopus (287) Google Scholar). GST-tagged SF2 and SF2 fragments (GST-ASF/SF21–119, GST-ASF/SF2120–194, and GST-ASF/SF2195–248) were expressed in E. coli strain BL21(DE) (Novagen) and purified on glutathione-agarose (Sigma) as previously described (25Kowalska-Loth B. Girstun A. Piekielko A. Staron K. Eur. J. Biochem. 2002; 269: 3504-3510Crossref PubMed Scopus (20) Google Scholar). GST-ASF/SF2195–248 was subjected to further purification on a heparin-agarose column after dilution in 50 mm Tris-HCl buffer, pH 8.0, containing 10 mm β-mercaptoethanol, 10% glycerol, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride. Elution was carried out in the same buffer with increasing NaCl concentrations (0.1–1 m). Purified polypeptides were concentrated on Biomax-5K columns (Millipore), and buffer was exchanged to 20 mm HEPES, pH 7.4, 50 mm NaCl, 75 mm KCl, 1 mm EDTA, 0.05% Triton X-100, 10% glycerol, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride. topo I Activity Assays—topo I DNA cleavage activity was assayed using a 32P-5′-end-labeled ds-oligonucleotide containing a single topo I binding/cleavage site, as previously reported (24Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Reaction mixtures (15 μl) were assembled on ice and contained 50 mm Tris-HCl, pH 8.0, 0.1 mm EDTA, 1 mm DTT, 0.01% Triton X-100, 20 μm CPT, 0.2 mg/ml bovine serum albumin, 2% glycerol, human topo I (0.2–0.27 pmol). Reaction was started by addition of 32P-5′-end-labeled ds-oligonucleotide (0.02–0.04 pmol, 0.5–1.1 × 106 dpm/pmol) and carried on for 10–20 min at 37 °C. After termination by addition of concentrated Laemmli buffer, cleavage complexes were separated by SDS-PAGE on 7.5% polyacrylamide gels and visualized by autoradiography. When present, affinity-purified PAR or ASF/SF2 were added to the reaction mixture at the amounts indicated in the figures. topo I kinase activity was assayed in the same buffer as indicated above in the presence of 10 mm MgCl2 and 1 μm γ-[32P]ATP (0.3 × 106 dpm/pmol), in a final volume of 15 μl. 0.2 pmol of yeast-expressed human recombinant topo I and 0.7–2.8 pmol of recombinant His-ASF/SF2 were used in each assay. After incubation at 37 °C for 20 min, reaction was stopped by adding concentrated Laemmli buffer; ASF/SF2 phosphorylation was visualized by autoradiography, after electrophoretic separation on 10% polyacrylamide gels (SDS-PAGE). ASF/SF2 phosphorylation by SRPK1 was assayed under the same conditions using 0.05 pmol of the recombinant kinase. Double DNA Cleavage/Protein Phosphorylation Assay—Recombinant human topo I (0.2 pmol) and recombinant His-ASF/SF-2 (2.8 pmol) were incubated at 37 °C for 20 min, in 50 mm Tris-HCl, pH 8.0, containing 0.1 mm EDTA, 1 mm DTT, 10 mm MgCl2, 0.01% Triton X-100, 20 μm CPT, 0.2 mg/ml bovine serum albumin, 2% glycerol, 32P-5′-end-labeled ds-oligonucleotide (0.034 pmol, 0.5–1.1 × 106 dpm/pmol), γ-[32P]ATP (1.5 pmol, 0.3 × 106 dpm/pmol). Reaction mixtures were assembled on ice and transferred into a pre-warmed water bath immediately after γ-[32P]ATP addition. Reaction products were analyzed by SDS-PAGE onto 4–15% polyacrylamide gradient gels, followed by autoradiography. When present, affinity-purified PAR was added to the reaction mixture at the amounts indicated in the figures. PAR Binding Assay—The PAR binding assay was carried on essentially as described by Panzeter et al. (26Panzeter P.L. Zweifel B. Malanga M. Waser S.H. Richard M. Althaus F.R. J. Biol. Chem. 1993; 268: 17662-17664Abstract Full Text PDF PubMed Google Scholar). Proteins were immobilized on nitrocellulose either by Western blotting after electrophoretic separation on polyacrylamide gels, or by slot blotting. Duplicate samples were either gold/Coomassie Blue stained for protein visualization or probed with [32P]PAR, washed with 10 mm Tris-HCl, 0.15 m NaCl, 0.05% (v/v) Tween 20, pH 7.4 (TBST), and analyzed by autoradiography. Where indicated, 0.5 m NaCl was added to the washing buffer (0.5 m NaCl-TBST). Protein Extracts—HeLa S3 cells were cultured in complete Dulbecco's modified Eagle's medium under standard culturing conditions and harvested at a subconfluent stage to be used for nuclear and cytoplasmic protein extract preparations, as previously described (27Blenn C. Althaus F.R. Malanga M. Biochem. J. 2006; 396: 419-429Crossref PubMed Scopus (92) Google Scholar). ASF/SF2 or histone phosphorylation by endogenous kinase(s) was assayed in the conditions described by Rossi et al. (6Rossi F. Labourier E. Forné T. Divita G. Derancourt J. Riou J.F. Antoine E. Cathala G. Brunel C. Tazi J. Nature. 1996; 381: 80-82Crossref PubMed Scopus (287) Google Scholar) using 0.5 μl of extract (1 μg of protein), in the presence or absence of PAR. Phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. Quantitative Analyses—Autoradiographic bands were quantified by scanning densitometry using the GS-710 Bio-Rad densitometer and the image analysis software QuantityOne (BioRad). Data are expressed as mean of at least three independent experiments ± S.D. Bioinformatics—Peer-reviewed literature was surveyed for comparative genome-wide studies of the transcriptomes of parp-1 knock-out mammalian cells and their wild-type counterparts, both under physiological conditions and after exposure to cytotoxic stimuli. Then, gene expression profiling data were matched against the Alternative Splicing Annotation Project II data base (28Kim N. Alekseyenko A.V. Roy M. Lee C. Nucleic Acids Res. 2007; 35: D93-D98Crossref PubMed Scopus (86) Google Scholar). Only genes coding for at least one expressed alternatively spliced isoform were taken as positive and expressed as the percentage of total analyzed genes. topo I Protein Kinase Activity Is Inhibited by PAR—In a reconstituted system consisting of PAR, topo I, and an oligonucleotide substrate, we first demonstrated that site-specific DNA cleavage by either yeast- or baculovirus-expressed recombinant human topo I is inhibited by PAR in a dose-dependent manner (Fig. 1A), as previously reported by us for topo I purified from human placenta (24Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). This inhibition is accompanied by the formation of a PAR·topo I complex (24Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar); degradation of PAR to monomeric ADP-ribose abolished the inhibition (Fig. 1C). To investigate whether PAR had any influence on topo I kinase activity, we performed an ASF/SF2 phosphorylation assay in the presence or absence of the polymer. topo I kinase targets the C-terminal RS domain of ASF/SF2 (29Labourier E. Rossi F. Gallouzi I.E. Allemand E. Divita G. Tazi J. Nucleic Acids Res. 1998; 26: 2955-2962Crossref PubMed Scopus (74) Google Scholar). His-tagged ASF/SF2, expressed in bacteria and purified by metal ion affinity chromatography, was used in this study. The recombinant protein migrated as a doublet in SDS-polyacrylamide gels (Fig. 2C); the shorter form is generated by proteolysis at the C terminus of the protein, which does not affect the capacity of the protein to be phosphorylated by SR-protein-specific kinases (29Labourier E. Rossi F. Gallouzi I.E. Allemand E. Divita G. Tazi J. Nucleic Acids Res. 1998; 26: 2955-2962Crossref PubMed Scopus (74) Google Scholar). Recombinant ASF/SF2 was phosphorylated by both topo I and the SR-protein kinase SRPK1 (Fig. 2, A and B). However, when PAR was present in the kinase assay reaction mixture, topo I-catalyzed ASF/SF2 phosphorylation was dramatically reduced (Fig. 2, B and E); the extent of inhibition was dependent on PAR concentration (Fig. 2, B and E). Thus, PAR appears to function as a negative regulator not only of the DNA cleavage (Fig. 1A) but also of the protein phosphorylation activity of topo I (Fig. 2, B and E). Neither PARP-1 nor PARP-2 in their native state affected topo I-catalyzed ASF/SF2 phosphorylation (Fig. 2D). Furthermore, the polymer at severalfold higher concentrations than that inhibitory on topo I had only modest consequences on ASF/SF2 phosphorylation by SRPK1 (Fig. 2, A and E), thus pointing at a topo I-specific PAR effect. Noteworthy, although both topo I and SRPK1 phosphorylate serine residues in the RS-domain of ASF/SF2, the reaction catalyzed by topo I has different specificity and kinetics from that of SRPK1 (29Labourier E. Rossi F. Gallouzi I.E. Allemand E. Divita G. Tazi J. Nucleic Acids Res. 1998; 26: 2955-2962Crossref PubMed Scopus (74) Google Scholar, 30Aubol B.E. Chakrabarti S. Ngo J. Shaffer J. Nolen B. Fu X.D. Ghosh G. Adams J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12601-12606Crossref PubMed Scopus (86) Google Scholar), implying the involvement of distinct mechanisms.FIGURE 2PAR inhibits ASF/SF2 phosphorylation. ASF/SF2 phosphorylation by either SRPK1 (0.05 pmol, A) or topo I (0.2 pmol, B) was assayed in the absence or presence of increasing amounts of PAR; alternatively, 0.18 pmol of native PARP-1 or 0.3 pmol of PARP-2 was added together with topo I and ASF/SF2 into the kinase assay mixture (D). Phosphorylated ASF/SF2 was visualized by autoradiography following SDS-PAGE (A, B, and D). Duplicate gels were stained with Coomassie Blue for protein visualization (C). Autoradiographic bands were quantified by scanning densitometry and data were expressed as percentage of enzyme activity in the absence of PAR (E); data from at least three independent experiments (mean ± S.D.) are shown. M, molecular mass markers; size of protein markers (kDa) are indicated by numbers on the left in C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PAR and ASF/SF2 Reciprocally Antagonize Their topo I Inhibitory Action—ASF/SF2 is known to inhibit DNA relaxation by topo I by interfering with the DNA cleavage step of the catalytic cycle (25Kowalska-Loth B. Girstun A. Piekielko A. Staron K. Eur. J. Biochem. 2002; 269: 3504-3510Crossref PubMed Scopus (20) Google Scholar, 31Andersen F.F. Tange T.O. Sinnathamby T. Olesen J.R. Andersen K.E. Westergaard O. Kjems J. Knudsen B.R. J. Mol. Biol. 2002; 322: 677-686Crossref PubMed Scopus (25) Google Scholar, 32Czubaty A. Girstun A. Kowalska-Loth B. Trzcinska A.M. Purta E. Winczura A. Grajkowski W. Staron K. Biochim. Biophys. Acta. 2005; 1749: 133-141Crossref PubMed Scopus (54) Google Scholar, 33Kowalska-Loth B. Girstun A. Trzcinska A.M. Piekielko-Witkowska A. Staron K. Biochem. Biophys. Res. Commun. 2005; 331: 398-403Crossref PubMed Scopus (10) Google Scholar). In Fig. 1B inhibition of topo I activity was achieved at an approximate topo I:ASF/SF2 molar ratio of 1:10. Because both PAR and ASF/SF2 are negative regulators of topo I-catalyzed DNA cleavage (Fig. 1), and ASF/SF2 phosphorylation is inhibited by PAR as well (Fig. 2), would PAR and ASF/SF2 together cause a complete silencing of topo I functions? To address this question, we set up an in vitro assay that allows simultaneous detection of topo I·DNA cleavage complex and phosphorylated ASF/SF2. Surprisingly, we observed a full restoration of topo I-catalyzed DNA cleavage while the inhibitory effect of PAR on topo I-dependent ASF/SF2 phosphorylation persisted (Fig. 3A). Reversal of topo I activity on DNA in the presence of both PAR and ASF/SF2 was clearly dependent on PAR concentration (Fig. 3A, right panel) and also occurred in the absence of ATP (Fig. 3B, left panel). It is noteworthy that cleavage complex formation by a N-terminally truncated form of topo I (ΔN-topo I, 70 kDa), which maintained full proficiency to relax DNA but had dramatically reduced protein kinase activity, was also inhibited by either PAR or ASF/SF2 individually (Fig. 3B, right panel). This is in agreement with our previous identification of PAR binding sites outside of the N-terminal domain of topo I (24Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Like full-length topo I, cleavage complex formation by ΔN-topo I was also restored in the presence of both PAR and ASF/SF2 (Fig. 3B, right panel). ASF/SF2 Is a Novel Member of the PAR-binding Protein Family—We next hypothesized that PAR could bind to ASF/SF2 and that the formation of a stable PAR·ASF/SF2 complex might prevent either of the two interaction partners from binding topo I; as a consequence, topo I would be able to express its DNA cleavage activity. PAR binding was assessed using recombinant ASF/SF2, either His-tagged or as a fusion protein with GST. To identify the specific domain(s) potentially involved in polymer binding, we constructed ASF/SF2 deletion mutants, each comprising only one of the ASF/SF2 functional domains (RRM1/RRM2/RS) (Fig. 4A). PAR binding assays revealed that ASF/SF2 does indeed bind PAR (Fig. 4, C and D). We identified the RRM1 and RS domains as potential targets of such interaction, whereas the RRM2 domain did not appear to possess any PAR binding activity (Fig. 4, C and D). Identical results were obtained both when proteins were first separated by SDS-PAGE and then transferred onto nitrocellulose by Western blotting (Fig. 4, B and D) and when native proteins where immobilized on the membrane by slot-blotting (Fig. 4C), before being probed with radioactive PAR. Importantly, ASF/SF2·PAR binding appeared to be considerably stronger than topo I-PAR interactions: nearly 90% topo I binding to PAR was destroyed by 0.5 m NaCl; in contrast, most ASF/SF2·PAR complexes resisted high salt treatment (Fig. 5A).FIGURE 5PAR binds to ASF/SF2 with higher affinity than to topo I. A, effect of high salt treatment on the stability of PAR-protein complexes. Recombinant proteins (5 pmol) were immobilized on nitrocellulose by slot blotting and either Gold stained (inset a) or incubated with [32P]PAR, followed by washes with either TBST or 0.5 m NaCl-TBST (insets b and c, respectively). PAR·protein complexes were visualized by autoradiography (insets b and c), and band intensities were quantified by scanning densitometry. Data are expressed as percentage of controls (no salt treatment) and represent mean values ± S.D. of at least three independent experiments. B, competition binding functional assay. topo I (0.2 pmol) was incubated with 5′-[32P]ds-oligonucleotide substrate and ASF/SF2 in the absence or presence of increasing amounts of PAR. topo I·DNA cleavage complexes were visualized by autoradiography following SDS-PAGE and quantified by scanning densitometry. Data shown represent the mean ± S.D. of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, the inhibitory effect of ASF/SF2 on DNA cleavage by a constant amount of topo I was titrated against increasing concentrations of PAR. We found that restoration of topo I DNA cleavage activity was strictly dependent on the relative amounts of the two negative effectors (Fig. 5B): 10 pmol of polymeric ADP-ribose were sufficient to f" @default.
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