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J Biol Chem, Vol. 273, Issue 41, 26261-26264, October 9, 1998
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From the Medizinische Poliklinik, University of
Wuerzburg, D-97070 Wuerzburg, Germany and § Department of
Structural and Molecular Biology, University of Aarhus,
DK-8000 Aarhus C, Denmark
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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DNA-topoisomerase I has been implied in RNA splicing because it catalyzes RNA strand transfer and activates serine/arginine-rich RNA-splicing factors by phosphorylation. Here, we demonstrate a direct interaction between topoisomerase I and pyrimidine tract binding protein-associated splicing factor (PSF), a cofactor of RNA splicing, which forms heterodimers with its smaller homolog, the nuclear RNA-binding protein of 54 kDa (p54). Topoisomerase I, PSF, and p54 copurified in a 1:1:1 ratio from human A431 cell nuclear extracts. Specific binding of topoisomerase I to PSF (but not p54) was demonstrated by coimmunoprecipitation and by far Western blotting, in which renatured blots were probed with biotinylated topoisomerase I. Chemical cross-linking of pure topoisomerase I revealed monomeric, dimeric, and trimeric enzyme forms, whereas in the presence of PSF/p54 the enzyme was cross-linked into complexes larger than homotrimers. When topoisomerase I was complexed with PSF/p54 it was 16-fold more active than the pure enzyme, which could be stimulated 5- and 16-fold by the addition of recombinant PSF or native PSF/p54, respectively. A physiological role of this stimulatory mechanism seems feasible, because topoisomerase I and PSF showed a patched colocalization in A431 cell nuclei, which varied with cell cycle.
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INTRODUCTION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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DNA-topoisomerase I alters the pitch of DNA double helices by cutting one DNA strand and allowing passage of the complementary strand through the transient nick (1). Thus, the enzyme releases torsional stress in the vicinity of replication forks (2) and transcription complexes (3). Topoisomerase I may also be involved in DNA recombination (4), DNA repair (5, 6), and chromosome formation (7), and it acts as a transcription factor (8) and a protein kinase (9) in a manner not involving DNA turnover. It is not clear how the enzyme is assigned to these multiple functions. Specific binding to various other nucleoproteins, such as RNA polymerase I (10), casein kinase II (11), nucleolin (12), p53 (13), and SV40 large tumor antigen (14) suggests that topoisomerase I may get recruited by protein-protein interactions to sites where its activity is required. On the assumption that such a regulatory mechanism is generally employed, we have searched for new proteins interacting with topoisomerase I and investigated their effects on catalytic DNA turnover by the enzyme.
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EXPERIMENTAL PROCEDURES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Recombinant Proteins-- Topoisomerase I was expressed in Saccharomyces cerevisiae and purified as described (15). Pyrimidine tract binding protein-associated splicing factor (PSF)1 was expressed in Escherichia coli and purified as described (16).
Protein Purifications from A431 Cells-- Topoisomerase I, PSF, and p54 (nuclear RNA-binding protein of 54 kDa) (17) were copurified from human A431 epidermoid cells (ATCC no. 1555) as follows. Nuclear extracts (5 ml) prepared from 109 cells in log growth as described (7) were digested with 3000 units of DNase I for 20 min at 20 °C and passed through a column (0.5 × 1 cm) of Ni-NTA-agarose (Qiagen, Hilden, Germany). The Ni-NTA-column was washed with 20 and eluted with 200 mM imidazole in buffer A (30 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM PMSF, 10% glycerol). The Ni-NTA eluate was passed through a Source-15Q column, adsorbed to a Source-15S column (both Pharmacia Biotech, Uppsala, Sweden), and finally eluted with 400 mM NaCl in buffer A. Topoisomerase I was separated from the PSF/p54 heterodimer by adding 1 M ammonium sulfate to the trimeric coeluate of Ni-NTA-agarose. The precipitate contained mainly PSF/p54 heterodimer, whereas topoisomerase I remained soluble. Precipitated PSF/p54 heterodimer was renatured and purified by gel permeation chromatography in the presence of 1 M NaCl using a Superdex 200 HR30/10 column (Pharmacia Biotech). Peak fractions were dialyzed against 15 mM HEPES, pH 7.5, 300 mM NaCl, 1 mM PMSF, and 50% glycerol. Native endogeneous topoisomerase I soluble in 1 M ammonium sulfate was adsorbed to phenyl-Sepharose (Pharmacia Biotech), eluted with 30 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM PMSF, 50% glycerol. Analytical gel permeation chromatography of coeluates of topoisomerase I, PSF, and p54 was carried out in the presence of 400 mM NaCl with or without 1 M urea using a Superdex 200 HR30/10 column.
Protein Chemistry--
For N-terminal microsequencing, protein
bands were cut out from SDS gels, subjected to BrCN cleavage,
re-electrophoresed, transferred to polyvinylidene difluoride membranes,
and sequenced from the blots by standard procedures using the automated
sequencing apparatus (Applied Biosystems model 476A). Protein
cross-linking reactions at 20 °C included 0.1-0.3 mg/ml protein (in
15 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM PMSF, 10% glycerol, and 10% Me2SO) and 0.5 mM SMCC (succinimidyl
4(N-maleinimidomethyl)-cyclohexane-1-carboxylate, Pierce)
and were stopped with 100 mM NH4Cl and
dithiothreitol after 20 min. Cross-linking products were separated in
4% SDS-polyacrylamide gels and analyzed by immunoblotting of
topoisomerase I. Recombinant human topoisomerase I was biotinylated
using D-biotinoyl-
-aminocaproic acid hydroxysuccinimidyl
ester (Boehringer Mannheim) without affecting catalytic activity.
Immunoassays-- Indirect immunofluorescence microscopy of topoisomerase I in A431 cells using human Scl-70 autoantibodies has been described (7). PSF was visualized similarly using a rabbit antiserum against a C-terminal peptide of human PSF (16), which was also employed for immunoblotting of PSF, whereas immunoblotting of topoisomerase I followed established procedures (7, 18) using the mouse monoclonal antibody NK 147 (18). Coimmunoprecipitation of topoisomerase I and PSF from chromatographic fractions (200 µl) was achieved by addition of Scl-70 autoantibodies (IgG fraction) to a final concentration of 1 µg/µl and 0.05% Nonidet P-40 and NaCl to a final concentration of 400 mM. After incubation for 1 h at 20 °C, 10 µl of protein A-Sepharose were added (Pharmacia Biotech, equilibrated with PBS containing 1% casein and 0.05% Nonidet P-40), and incubation continued for 1 h at 20 °C. Finally, protein A-Sepharose was sedimented, washed, and eluted with 2% SDS, and eluates were analyzed by immunoblotting with PSF antibodies. For far Western blotting, the blots were renatured for 12 h at 20 °C with PBS containing 0.1% Tween 20 (PBS-Tween) and 5% fat-free milk. After renaturation the blots were incubated for 1 h at 20 °C with the biotinylated enzyme (10 ng/ml in PBS-Tween containing 2% bovine serum albumin). After washing (PBS-Tween + 0.5 M NaCl) bound enzyme was detected with alkaline phosphatase-conjugated streptavidin (AvidixTM) and a chemiluminescent substrate (CSPDTM, both Tropix, Serva, Heidelberg, Germany).
Catalytic Assays-- Kinetic determinations of specific topoisomerase I activities were performed with 2.5 ng of pure topoisomerase I or equivalent amounts of the enzyme contained in other preparations and 0.3 µg of supercoiled pUC18 DNA. Reactions were carried out in a final volume of 20 µl of assay buffer (10 mM BisTris propane, pH 7.9, 50 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 0.3 µg/µl bovine serum albumin, and 0.5 mM dithiothreitol) at 37 °C and were stopped by addition of 1% SDS at the time points indicated. Samples were digested with proteinase K (0.1 mg/ml, 37 °C, 30 min). Relaxed and supercoiled plasmid forms were separated by submarine agarose gel electrophoresis, stained with 0.5 µg/ml ethidium bromide, visualized by UV transillumination, and photographed. Images were scanned, and the relative amounts of relaxed DNA were determined using the image analysis software NIH Image v.1.61. One unit of topoisomerase I activity was defined as the amount of enzyme required for relaxation of 150 ng of supercoiled pUC18 DNA within 30 min. Concentrations of pure recombinant topoisomerase I and pure recombinant human PSF were determined according to Bradford and served as a standard for determining the respective protein concentrations in crude nuclear extracts, preparations of PSF/p54 heterodimers, and trimeric coeluates by comparative immunoblotting. To test the effect of PSF or PSF/p54 on topoisomerase I activity, 2.5 ng of pure enzyme or an equivalent amount of topoisomerase I-PSF/p54 coeluate was preincubated (15 min, 37 °C) with 120 ng of pure recombinant human PSF or an equivalent amount of isolated native PSF/p54 dimer in a final volume of 20 µl of assay buffer.
Statistics--
Quantitative results are represented as mean
values ± standard error of the mean (standard deviation divided
by square root of n 
1) of three or more independent
measurements carried out independently on different days. Fluorescent
images of cells are representative of the whole cell population
inspected in at least 10 separate fields of view.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Interaction of Topoisomerase I to PSF and p54-- Topoisomerase I can be purified from ammonium sulfate precipitates of human nuclear extracts by a single step of nickel affinity chromatography (9). By the same principle, we copurified here a set of three proteins (54, 100, and 115 kDa) from A431 cell nuclear extracts not subjected to high ammonium sulfate concentrations before chromatography (Fig. 1a, lanes 1-3). Copurification in a constant stoichiometric ratio of 1:1:1 was conserved during several other chromatographic procedures including anion exchange (Fig. 1a, lanes 4 and 5), cation exchange (Fig. 1a, lanes 6 and 7), and gel permeation chromatography (Fig. 1b, top) but was disrupted by 1 M urea (Fig. 1a, lanes 8 and 9; Fig. 1b, bottom) or 1 M ammonium sulfate (Fig. 4a, lanes 2 and 4), suggesting complexation of these three proteins rather than similar matrix interactions. The 100-kDa protein was identified as topoisomerase I by immunoblotting (Fig. 2a, lane 5), whereas the two other proteins were identified by N-terminal microsequencing. From the 115-kDa protein three peptide sequences identical to amino acid residues 4-21, 214-219, and 685-693 of human PSF (16) were obtained. The identity of PSF was subsequently confirmed by immunoblotting (Fig. 2a, lane 6). From the 54-kDa protein three peptide sequences identical to amino acid residues 4-14, 90-120, and 443-466 of human nuclear RNA-binding protein p54 (17) were obtained. PSF and topoisomerase I could be coimmunoprecipitated with topoisomerase I antibodies (Fig. 2b, lane 3), and a specific binding of pure recombinant human topoisomerase I to PSF but not to p54 could be demonstrated by far Western blotting, probing renatured Western blots of crude nuclear extracts (Fig. 2a, lane 3) or coeluates of topoisomerase I, PSF, and p54 (Fig. 2b, lane 4) with biotinylated topoisomerase I. From the experiment in Fig. 2a, lanes 3 and 4, it became apparent that topoisomerase I also binds strongly to itself, which suggests multimerization of the enzyme. Treatment of pure recombinant human topoisomerase I (Fig. 2c, lane 5) with SMCC stabilized fractions of the enzyme in dimeric (30%) or trimeric (10%) complexes (Fig. 2c, lane 1). When coeluates containing topoisomerase I, PSF, and p54 but no other proteins (Fig. 2c, lane 6) were treated with SMCC, similar fractions of di- and trimeric topoisomerase I were obtained, but in addition, the enzyme became cross-linked into complexes much larger than homotrimers (Fig. 2c, lane 2). These data indicate that human topoisomerase I binds PSF, which in turn is associated with p54.
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Colocalization of Topoisomerase I and PSF in A431 Cells-- To determine whether such complexes could also assemble in the cell, we double stained topoisomerase I and PSF in human A431 cells and compared their cellular localization patterns by indirect immunofluorescence microscopy (Fig. 3). During most of mitosis the two proteins were separated because PSF diffused into the cytosol in prometaphase (Fig. 3a), remained excluded from the chromatin during metaphase (Fig. 3b) and early anaphase (Fig. 3c), and started to reassociate with the DNA in late anaphase (Fig. 3d) and telophase (Fig. 3e). Topoisomerase I, on the other hand, remained entirely chromatin-bound during all these mitotic steps. In most interphase cells both proteins had a diffuse nuclear distribution (in the case of PSF strictly excluding the nucleoli) and showed extended patches of colocalization in the extranucleolar nucleoplasm (Fig. 3f, yellow patches), whereas in a minor fraction (5-10%) of interphase nuclei the two proteins showed grainy and inhomogeneous patterns and did not colocalize but, on the contrary, appeared to be compartmentalized away from each other (Fig. 3g). These observations suggest that interactions between topoisomerase I and PSF can occur in the cell and might be governed by cell cycle-related redistribution and compartmentalization.
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Stimulation of Topoisomerase I by PSF-- Initially, we observed that topoisomerase I of human A431 cells was 8-fold more active in the context of a crude nuclear extract (Table I, line 8) than in the purified state (Table I, line 4). The pure enzyme of A431 cells had a specific activity of 2.9 × 103 units/µg similar to pure recombinant human topoisomerase I (Table I, line 1) and Ref. 19, whereas in the copurified complex with PSF/p54 topoisomerase I was even more active than in the nuclear extract (Table I, line 7). Thus, we assumed that the decrease in enzyme activity upon purification could be because of the removal of PSF/p54. To test this assumption, we preincubated pure recombinant (Fig. 4a, lane 1) or endogeneous topoisomerase I (Fig. 4a, lane 2) with pure recombinant PSF (Fig. 4a, lane 3) or isolated native PSF/p54 dimer (Fig. 4a, lane 4) and compared it to the copurified topoisomerase I-PSF/p54 complex (Fig. 4a, lane 5). Such an experiment is shown in Fig. 4b. Quantitative results are summarized in Table I. The isolated native PSF/p54 dimer, which did not have DNA relaxation activity of its own (Fig. 4b), stimulated the activity of purified endogeneous topoisomerase I at least 16-fold (thus entirely restituting the activity of the trimeric complex), whereas it had a slightly lesser effect on recombinant human topoisomerase I. Recombinant human PSF also stimulated purified topoisomerase I but was 3 times less effective than the native PSF/p54 dimer. The activity of topoisomerase I in the copurified equimolar complex with PSF/p54 was insignificantly increased by preincubation with a 50-fold molar excess of recombinant human PSF or isolated native PSF/p54 dimer (Table I), suggesting that the stimulatory effect was saturated at equimolar ratios of the three proteins. In summary, these data show that PSF/p54 is a strong stimulator of topoisomerase I in human A431 cells.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Human topoisomerase I is involved in the activation of serine/arginine-rich cofactors of RNA splicing, either by an endogenous protein kinase activity (9) or by physical association with such an enzyme (21). Here, we report on a direct interaction of topoisomerase I and another RNA-splicing factor. We show that human topoisomerase I binds PSF, an essential cofactor of the second step of RNA splicing (16), and is indirectly (via PSF (22, 23)) associated with p54, a PSF homolog of unknown function (17). Furthermore, we show that the PSF/p54 dimer has pronounced stimulatory effect on DNA catalysis by topoisomerase I in vitro. Thus, PSF/p54 could be a regulator of topoisomerase I, independent of and in addition to its functions in RNA splicing. However, because PSF is a well known RNA-splicing factor, it is also tempting to speculate that the observed interaction between topoisomerase I and PSF/p54 reflects a function of topoisomerase I in RNA splicing.
In contrast to stimulation of topoisomerase I by histone H1 and high mobility group proteins (11), which probably reflect an indirect DNA-mediated effect, stimulation of topoisomerase I by PSF/p54 was seen with equimolar complexes of these proteins and could not be significantly increased by further addition of the stimulatory proteins at a 50-fold molar excess. Thus, it seems to be mostly because of a direct protein-protein interaction. As we were not able to separate the native PSF/p54 dimer, we could not directly assess the individual contribution of p54 to this stimulatory effect. We observed that recombinant PSF alone was not as effective as the native PSF/p54 dimer. This could be because of the lack of p54 but could equally well be because of inappropriate folding of the recombinant PSF or lack of posttranslational modifications. It should be noted that endogeneous PSF of A431 cells had a slightly different migration in a SDS gel than recombinant PSF produced in E. coli (Fig. 4a, lanes 3 and 4).
Partial colocalization of PSF and topoisomerase I in interphase nuclei of human A431 cells (Fig. 3, e-g) suggests that complexes of these proteins could assemble in the cell in a manner regulated by compartmentalization. We have not yet obtained direct evidence that such interactions actually serve the regulation of topoisomerase I in a living cell. However, it should be noted that the complete dissociation of topoisomerase I and PSF during early mitosis shown in Fig. 3, a-d, coincides with a 4-fold drop in topoisomerase I activity extractable from mitotic A431 cells (7).
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ACKNOWLEDGEMENTS |
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Rabbit antibodies and the clone of human PSF were kindly supplied by Dr. J. G. Patton, Department of Molecular Biology, Vanderbilt University, Nashville TN. We gratefully acknowledge the excellent technical assistance of Claudia Volff. We are also grateful to Dr. Jørgen Kjems, Department of Structural and Molecular Biology, University of Aarhus, Denmark, for helpful discussions.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 172, B12, and Bo 910/2-1), the Danish Cancer Society (Grant 97-100-32), the Danish Center for Human Genome Research, and the Danish Center for Molecular Gerontology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of an educational stipend by the Deutsche Krebshilfe, Dr. Mildred Scheel-Stiftung. To whom correspondence should be addressed: Medizinische Poliklinik der Universität, Klinikstrasse 6-8, D 97070 Wuerzburg, Germany. Tel.: 49-931-201-7008; Fax: 49-931-201-7120; E-mail: F.Boege{at}medizin.uni-wuerzburg.de.
The abbreviations used are: PSF, pyrimidine tract binding protein-associated splicing factor (16)BisTris propane, (1,3-bis[tris(hydroxymethyl)-methylamino]propane)Me2SO, dimethyl sulfoxideNTA, nitrilotriacetic acidPBS, phosphate-buffered saline solutionPMSF, phenylmethylsulfonyl fluoridep54, nuclear RNA-binding protein of 54 kDa (17)SMCC, succinimidyl 4(N-maleinimidomethyl)- cyclohexane-1-carboxylate.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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