(Received for publication, August 23, 1995; and in revised form, November 17, 1995)
From the
DNA topoisomerase I (topo I) is involved in the regulation of DNA supercoiling, gene transcription, and rDNA recombination. However, little is known about interactions between topo I and other nuclear proteins. We used affinity chromatography with a topo I fusion protein to screen U-937 leukemic cell extracts and have identified nucleolin as a topo I-binding protein. Coimmunoprecipitation and other studies demonstrate that the interaction between topo I and nucleolin is direct. Furthermore, deletion analyses have identified the 166-210-amino acid region of topo I as sufficient for the interaction with nucleolin. Since nucleolin has been implicated in nuclear transport and in a variety of transcriptional processes, the interaction with topo I may relate to the cellular localization of topo I or to the known role of this topoisomerase in transcription.
Eukaryotic type I topoisomerases are capable of relaxing both
negatively and positively supercoiled
DNA(1, 2, 3) . This function is believed to
be important in cellular processes requiring access to DNA, such as
transcription and replication, and inhibitors of topo I ()block these
events(4, 5, 6, 7, 8) .
However, yeast mutants lacking the enzyme exhibit nearly normal growth,
and it has been proposed that other cellular topoisomerases may
compensate for the lack of topo I(9) . The finding that yeast
topo I mutants exhibit an increase in rDNA recombination has supported
an irreplaceable role for topo I in maintaining the integrity of the
rDNA locus(10) . Furthermore, several studies have indicated
that topo I is located predominately in the
nucleolus(11, 12, 13, 14) , although
the mechanisms related to nucleolar localization of topo I and its
unique effects on rDNA are unclear.
In the present studies, we used affinity chromatography with an immobilized fusion protein to identify proteins interacting with topo I. We report that nucleolin, a major nucleolar protein, exhibits binding to topo I in both affinity chromatography and coimmunoprecipitation experiments. A region in the amino terminus of topo I has been identified that is sufficient for the interaction with nucleolin.
For isolation of topo-I-binding
proteins from crude cell extracts, U-937 leukemic cells in exponential
growth were collected by centrifugation and washed with PBS. After
resuspension in lysis buffer (PBS containing 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10
µg/ml leupeptin, 0.1 mM pepstatin), cells were lysed in a
Dounce homogenizer and then subjected to brief sonication. After
addition of an equal volume of PBS containing 0.2% Tween, the extracts
were centrifuged at 15,000 g for 10 min. The
supernatants were added to pre-equilibrated GST-glutathione-Sepharose
beads. The flow through and a 1 bed volume wash with PBS, 0.2% Tween
were collected and added to pre-equilibrated GST-fusion
protein-Sepharose beads. The resulting slurry was incubated for 1 h
with rotation, and the beads were then washed twice with 10 bed volumes
of PBS, 0.2% Tween. Washing continued with PBS until the A
of the eluate was <0.01. Bound proteins
were eluted with glycine-HCl, and the eluates were neutralized with
K
HPO
. The eluates were concentrated, and the
buffer was exchanged with PBS, 20% glycerol with the use of
Centricon-30 ultrafiltration devices (Amicon). Aliquots of the
concentrated eluates were analyzed by SDS-PAGE, with proteins
visualized by Coomassie Blue or silver staining.
Figure 1: Analysis of topo I-binding proteins in U-937 cell extracts. Crude cell extracts were subjected to affinity chromatography using GST or GST-topo I-Sepharose beads, as described under ``Materials and Methods.'' Bound proteins were eluted and analyzed by polyacrylamide gel electrophoresis, transfer to polyvinylidene difluoride, and Coomassie Blue staining. A photocopy of a representative stained membrane is shown. Lanes are labeled according to the matrix used for chromatography, except for the lane labeled PBS, which represents an experiment with GST-topo I beads where PBS was substituted for cell extract. Arrows represent bands that were removed for amino-terminal sequencing.
Figure 2: Immunoblotting of topo I-binding proteins with nucleolin antibody. Cell extracts were subjected to affinity chromatography as described in the legend to Fig. 1. Bound proteins were analyzed by immunoblotting with a monoclonal nucleolin antibody, using a chemiluminescent technique. Lanes are labeled according to the matrix used for chromatography, with the exception of the third lane, which represents direct immunoblotting of U-937 cell extract.
To confirm a potential interaction between nucleolin and topo I, we performed coimmunoprecipitation assays. Attempts to immunoprecipitate topo I with a polyclonal antibody from scleroderma patients (22) were unsuccessful (data not shown). However, immunoprecipitates prepared with a monoclonal anti-nucleolin antibody contained proteins of approximately 75 and 67 kDa, which were reactive with the topo I antibody (Fig. 3). Proteins of identical electrophoretic mobility were identified by this antibody in cell extracts, indicating that they represent topoisomerase I fragments. The detection of degraded forms of topo I is consistent with previous results using U-937 cells(15) . In contrast to the results obtained with the anti-nucleolin antibody, topo I was not found in control immunoprecipitates using rabbit pre-immune serum (Fig. 3). Thus, binding of nucleolin to topo I is detectable using both affinity chromatography and coimmunoprecipitation.
Figure 3: Topo I coimmunoprecipitates with nucleolin. U-937 cells were lysed by sonication in a buffer containing 1% Nonidet P-40. Aliquots of the soluble cell extract were either loaded directly onto a polyacrylamide gel (first lane) or incubated with either a monoclonal nucleolin antibody or preimmune rabbit serum (PIRS), followed by incubation with protein A-linked Sepharose beads. After extensive washing, bound proteins were released by boiling in loading buffer, and the samples were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with a polyclonal human topo I antibody.
Figure 4: Binding of nucleolin (Nucn.) to topo I does not involve stoichiometric quantities of other proteins or DNA. A, 5 µg of purified nucleolin were added to 25 µl of covalently linked GST-topo I beads. After washing, bound proteins were released by boiling and analyzed by electrophoresis and silver staining. For comparison, the first lane contains 1 µg of nucleolin alone. B, 2 µg of purified nucleolin were added to 25-µl aliquots of covalently linked GST-topo I beads in a buffer of PBS and 0.2% Tween. In addition, certain reactions contained either DNase I (1 µg/ml) or ethidium bromide (50 or 100 µg/ml), as indicated. After extensive washing, bound proteins were released from the beads by boiling and analyzed by SDS-PAGE and immunoblotting with nucleolin antibody.
Since both nucleolin and topo I are capable of binding DNA, experiments were performed to determine whether the interaction between nucleolin and topo I involves intermediary DNA. Inclusion of DNase in the binding buffer did not abolish the binding of nucleolin to topo I (Fig. 4B). Similar experiments were performed with ethidium bromide, which is known to abrogate DNA-dependent protein interactions(23) . Inclusion of up to 100 µg/ml ethidium bromide in the binding buffer did not affect the binding of nucleolin to topo I (Fig. 4B). These results suggest that DNA is not required for the interaction between nucleolin and topo I. Similar experiments with RNase indicated that RNA is also not involved in this interaction (data not shown).
Figure 5: Analysis of the binding of purified nucleolin to topo I fragments. A, expression of topo I deletion mutants. Aliquots of the indicated purified GST-topo I proteins were subjected to SDS-polyacrylamide gel electrophoresis and Coomassie staining. B, analysis of nucleolin binding to topo I fragments. 2 µg of purified nucleolin were added to glutathione-Sepharose beads containing the indicated fragment of topo I. After extensive washing, the beads were boiled in loading buffer, and released proteins were analyzed by immunoblotting with nucleolin antibody. C, schematic diagram of the results of the binding experiments. The relative levels of nucleolin binding to the topo I fragments are indicated by + signs.
The purified GST-topo I fragments were covalently linked to beads and assayed for nucleolin binding. The results indicate that the first 210 amino acids of topo I are necessary for nucleolin binding and that the first 250 amino acids are sufficient for this interaction (Fig. 5, B and C). The critical residues involved in nucleolin binding appear to be contained in the region 166-210, since a fusion protein containing this region is capable of binding nucleolin, whereas fragments flanking this region are not (Fig. 5, B and C).
Figure 6: Effect of nucleolin (Nucn.) on topo I plasmid relaxation activity. The ability of topo I to relax supercoiled plasmid DNA was analyzed by agarose gel electrophoresis and ethidium bromide staining. From left to right, the lanes represent the following reactions: 1) supercoiled plasmid alone, 2) plasmid + 60 ng of GST-topo I, 3-6) plasmid + 60 ng of GST-topo I + 1, 2, 3, or 4 ng of nucleolin, respectively, and 7) plasmid + 4 ng of nucleolin.
The primary cellular function of eukaryotic topoisomerase I is believed to be regulation of DNA supercoiling. Indeed, type I topoisomerases have been found in nearly every prokaryotic and eukaryotic cell examined and are also found in mitochondria and viruses (2) . We sought to gain further insights into the cellular role and regulation of topo I by identifying proteins that interact with this enzyme. Using affinity chromatography and immunoprecipitation, we have identified the 100-kDa nucleolar protein nucleolin as a topo I-binding protein. Previous immunohistochemical studies have indicated that nucleolin and topo I are located in similar regions of the nucleolus (25) . Furthermore, several studies have implicated topo I in the maintenance of both nucleolar structure and function(6, 25, 26, 27, 28) . The importance of topo I in the nucleolus is highlighted by the finding that yeast topo I deletion mutants exhibit an increase in rDNA recombination(10) .
Our data suggest that the nucleolin-topo I interaction is direct and does not require nucleic acid or other proteins. By deletion analysis we have determined that the nucleolin binding site in topo I is contained within the first 210 amino acids and that the 166-210 region is sufficient for this binding. This region consists of mostly charged residues, with a slight predominance of basic amino acids and a predicted pI of 8.7. These characteristics are consistent with the finding that nucleolin binding to topo I is impaired at pH 10.0 and in solutions of greater than 0.25 M NaCl (data not shown). Although nucleolin is an acidic protein, charge alone does not appear to be sufficient to determine binding of nucleolin by topo I peptides, since other basic fragments, e.g. the 140-167 and 1-139 regions (predicted pI values of 8.3 and 9.2, respectively) do not bind nucleolin. Similar results have been reported in analyses of the binding of basic peptides containing nuclear localization sequences (NLSs) by the nucleolar protein B23, in which charge alone was found to be insufficient to determine binding(29) . Further analysis of the 166-210 region of human topo I indicates that while it is highly conserved among higher eukaryotes, it is only partially conserved in yeast (Table 3). This suggests that the nucleolin-topo I interaction may be species specific. Indeed, to our knowledge nucleolin has been identified only in higher eukaryotes.
The finding that nucleolin appears to have little effect on topo I catalytic activity is consistent with results indicating that deletion of the first 209 amino acids of topo I (and thus the nucleolin binding site) does not impair plasmid relaxation activity (data not shown). Moreover, a 67-kDa carboxyl fragment of human topo I (30) and a 141-201 deletion mutant of Saccharomyces cerevisiae topo I (31) have both been shown to possess a catalytic activity similar to that of the intact protein. If nucleolin binding does not have a significant effect on topo I catalytic activity, then what is the purpose of this interaction? Although further study is required to answer this question, several hypotheses may be generated in view of the known functions of nucleolin. This protein constitutes approximately 5% of the total nucleolar protein and has been implicated in nucleolar organization, including rRNA synthesis and shuttling of nucleolar proteins from the cytoplasm to the nucleolus (32, 33, 34, 35) . In addition, the central region of nucleolin contains four consensus RNA binding motifs, which are likely involved in rRNA processing or ribosome assembly(18, 32) . Nucleolin has also been assigned more general nuclear roles, including involvement in nuclear transport(34) , chromatin decondensation(33) , and in the formation of the nuclear matrix(36) . Furthermore, nucleolin has been shown to be a transcriptional repressor for the alpha-1 acid glycoprotein gene(37) . Taken together, these findings indicate that nucleolin is involved in several aspects of nuclear structure and transcriptional regulation.
Since topo I is likely involved in regulation of supercoiling during transcription, one possible role for nucleolin binding is to target topo I to sites of transcription. This hypothesis is consistent with prior work indicating interactions between topo I and other transcription proteins, such as RNA polymerase I(38) , TATA-binding protein(39) , and high mobility group proteins(40) . Alternatively, nucleolin binding to topo I may be the mechanism by which topo I enters the nucleus. Indeed, deletion of the 140-230 region in yeast topo I results in loss of the nuclear localization of the protein, indicating that NLSs reside in this region(31) . Since the corresponding region in the human enzyme includes the 166-210 segment, it is possible that nucleolin binding is required for the nuclear localization of the human enzyme. In this regard, it is important to note that while the 166-210 region of topo I does contain a putative bipartite NLS(24) , the 140-167 and 1-139 regions each contain a potential bipartite NLS and do not bind nucleolin. Experiments with epitope-tagged topo I fragments should allow determination of whether the 166-210 region of topo I is necessary and/or sufficient for the nuclear and nucleolar localization of topo I.