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Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen
Introduction
Materials And Methods
Yeast two-hybrid assay and yeast and bacterial expression plasmids
Yeast lysate preparation
Preparation of immobilized Top1 and in vitro binding assays
Co-immunoprecipitation
Results
An SV40 T antigen fragment binds an N-terminal fragment of human Top1 invitro
SV40 T antigen interacts with the N-terminus of human Top1 in yeast
Identification of SV40 T antigen-binding regions in human Top1
Discussion
Acknowledgements
References
Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen
ABSTRACT
INTRODUCTION
Four human DNA topoisomerases have now been identified: topoisomerase I, topoisomerase II[alpha] and [beta] and topoisomerase III (1). Studies in yeast have suggested certain roles for the yeast homologues of these human proteins, including yeast topoisomerase I, II and III (2-8). With regard to yeast topoisomerase I, although this protein is not required for viability, yeast cells lacking topoisomerase I exhibit slower growth and an increase in ribosomal DNA recombination (5,9,10). In contrast with the yeast protein, the human topoisomerase I protein (Top1) is required for viability (11). Microinjection studies using antibodies that inhibit the enzymatic activity of topoisomerase I suggest that this activity is required for transcription by RNA polymerases I and II (12). Intriguingly, several new functions have been ascribed recently to Top1. These include a role in the suppression and activation of transcription by RNA polymerase II (13,14) and a role in phosphorylation of certain splicing factors (15). Top1 is also known to be an important antineoplastic drug target; the plant alkaloid camptothecin specifically targets this enzyme and two analogues have recently been approved for the treatment of colon and ovarian cancers (16,17). Camptothecins are known to bind and stabilize a Top1-DNA reaction intermediate, resulting in protein-bound single strand breaks in DNA (18). However, the formation of these complexes is not sufficient to cause cell death, and secondary events, such as the formation of DNA double strand breaks, are believed necessary to convert the protein-linked single strand breaks into a lethal injury (19-21). Although DNA double strand breaks may result from collisions of replication forks with camptothecin-Top1-DNA ternary complexes (22,23), camptothecin is also cytotoxic for non-replicating cells, with the mechanisms underlying these effects not yet clear (24).
In order to gain information regarding the cellular role of Top1 and perhaps mechanisms by which camptothecin kills both replicating and non-replicating cells, we have sought to identify Top1-interacting proteins. In previous work we identified an interaction between the N-terminus of Top1 and nucleolin (25), which is a DNA and RNA helicase (26). We have continued to identify proteins that interact with the N-terminus of Top1 and now report binding of this region by the Simian Virus 40 (SV40) T antigen.
MATERIALS AND METHODS
Yeast two-hybrid assay and yeast and bacterial expression plasmids
In order to generate a plasmid-based [beta]-galactosidase reporter system for use in a yeast two-hybrid screen, we generated yeast strain Y199 by selection of 5-fluoroorotic acid-resistant colonies of strain Y190 (Clontech, Palo Alto, CA) (27) that were incapable of growing on uracil-deficient media. This selection allowed transformation of Y199 with the plasmid pRY131, kindly provided by Dr Mark Ptashne, which contains a 2 µM replicator and expresses the [beta]-galactosidase gene under the control of the GAL1 promoter (28). For two-hybrid screening, yeast transformations were performed using lithium acetate and appropriate selective media. After 3-5 days of growth, [beta]-galactosidase activity was assayed using a filter lift technique and 5-bromo-4-chloro-3-indolyl-[beta]-d-galactopyranoside as described previously (29).
The plasmids pAS2-1 and pACT2 (Clontech) were used to express the GAL4 DNA-binding domain (BD) and the GAL4 activation domain (AD) proteins, respectively. Similarly, plasmid pTD1-1 (Clontech) was used to express a peptide consisting of amino acids 84-708 of the SV40 large T antigen fused to AD and plasmid pVA3-1 (Clontech) was used to express the amino acids 72-390 of murine p53 fused to BD. A yeast plasmid expressing the N-terminus of human Top1 linked to BD was constructed using PCR with primers designed to amplify from plasmid pGEX-TOP1 (30) the region of the human topoisomerase I cDNA coding for amino acids 2-250. The BamHI and PstIsites in pGBT9 (Clontech) were used to insert the amplified cDNA, yielding the PGBT9-Atop1 plasmid. Subsequently, the BamHI-PstI insert from this plasmid was placed into the pAS2-1 vector to allow higher levels of expression than with the pGBT9 vector. The resultant pAS2-Atop1 vector was used to express the BD-Atop1 protein in all of the experiments described here.
In order to obtain a recombinant source of nucleolin, we constructed a plasmid expressing a GST-nucleolin fusion protein. We used yeast to express the protein because of difficulties with construction of a prokaryotic vector. A nucleolin cDNA was obtained using human U-937 cell total RNA and RT-PCR with primers designed to amplify the 2.1 kb coding region of nucleolin (primer sequences available upon request). The resultant PCR product was inserted into the pACT2 vector (yielding the pACT-Nuc plasmid) using BamHI/EcoRI digestion. Subsequently, a yeast vector expressing GST-nucleolin was generated by insertion of a SmaI-EcoRI fragment from pACT-Nuc into the pKG vector kindly provided by Dr Robert Deschenes (31). The resultant pKG-Nuc plasmid was subsequently used to transform the yeast strain JCW25 [MATa, ura3-52, leu2-[Delta]1, trp1-[Delta]63(GAL3), his3-[Delta]200], obtained from Dr Marc Gartenberg (32).
Bacterial plasmids expressing human Top1 fragments as GST fusion proteins have been described previously (25). In addition, a plasmid expressing a GST fusion protein containing amino acids 383-765 of human Top1 was constructed using the pGEX-TOP1 plasmid (30) and CelII/BamHI digestion. Originally, a plasmid expressing a 251-765 fragment of Top1 was expected from this strategy and screening of recombinant clones by glutathione affinity chromatography yielded a clone expressing a 70 Mr peptide, similar to the expected 87 Mr of a GST fusion protein containing the 251-765 region of Top1. However, sequencing of this recombinant clone indicated that the clone actually expressed a 383-765 fragment of Top1 (apparently the result of BamHI star activity).
Yeast lysate preparation
Yeast were grown at 30°C in appropriate synthetic dropout media to an OD600 of 0.7-0.9, then pelleted and resuspended in cold RIPA buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and 1% Na-deoxycholate) with protease inhibitors (1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.5 µg/ml leupeptin and 1 µg/ml pepstatin). The suspension was vortexed with glass beads for 5 min at 4°C and the resultant lysate cleared by centrifugation at 1000 g for 5 min. The supernatant was removed and cleared of remaining precipitate by centrifugation at 14 000 g for 5 min. The resultant crude lysate was used for subsequent experiments, with protein concentration determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).
To obtain purified GST-nucleolin expressed from yeast, cells were first grown to an OD600 of 0.8 in media lacking uracil and dextrose, but containing 2% raffinose. Expression of GST-nucleolin was induced by addition of 2% galactose. After additional growth for 2 h, the yeast cells were pelleted and lysates prepared as described above. Affinity purification of GST-nucleolin was performed using glutathione-Sepharose beads as described (25). The purified GST-nucleolin protein was >90% pure as assessed by silver staining and was immunoreactive with a monoclonal anti-nucleolin antibody (data not shown) (33).
Preparation of immobilized Top1 and in vitro binding assays
Human Top1 peptide fragments were covalently linked to glutathione-Sepharose beads using the bifunctional imidoester dimethylpimelimidate dihydrochloride as described (25). Using the Top1-linked beads as bait, binding assays were performed using both crude yeast lysates and purified T antigen. The purified T antigen (kindly provided by Dr Steven Brill) was produced using a recombinant baculovirus and an immunoaffinity purification procedure as described (34-36). In the binding experiments, [sim]100 µg of yeast lysate protein or 1 µg of purified T antigen was added to the Top1-linked beads (30 µl of a 50% slurry for lysates, 10 µl for purified T antigen) in 1 ml PBS/0.2% Tween-20 containing protease inhibitors. In certain incubations, ethidium bromide was added to 100 µg/ml. In other experiments, purified recombinant GST-nucleolin was mixed with purified T antigen (at various molar ratios), followed by the addition of Top1-linked beads. Binding mixtures were rocked at 4°C for 1 h, briefly spun at 500 g and the supernatant removed. The beads were then washed three times with PBS/0.2% Tween-20 and twice with PBS. The pellet was resuspended in 10 µl of 2× SDS-PAGE sample dye [125 mM Tris-HCl, pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol, 0.01% (w/v) phenol red], incubated for 10 min at 95°C, subjected to brief centrifugation and the supernatant loaded onto a 10% SDS-PAGE gel. Bound proteins were visualized by silver staining or by immunoblotting with a monoclonal antibody recognizing the GAL4 activation domain (Clontech) and an enhanced chemiluminescent technique as described (25).
Figure 1. SV40 T antigen binds an N-terminal fragment of human topoisomerase I in vitro. Crude lysates were prepared from yeast strains expressing a GAL4 activation domain-T antigen fusion protein (AD-T Ag) or the GAL4 activation domain peptide alone (AD). The lysates were either loaded directly onto a 10% polyacrylamide gel (lanes 1 and 3), or incubated with Sepharose beads covalently linked to either a GST fusion protein containing an N-terminal fragment (amino acids 2-250) of human topoisomerase I (Atop1; lanes 2, 5 and 6), or the GST protein alone (GST; lane 4). After extensive washing, the beads were boiled in SDS-PAGE loading dye and subjected to electrophoresis and immunoblotting using an antibody recognizing the GAL4 activation domain. As indicated, certain incubations contained 100 µg/ml ethidium bromide (EtBr) to disrupt DNA-protein interactions. The migration of the GAL4 activation domain-T antigen fusion protein is indicated by the arrow.
Co-immunoprecipitation
Yeast strain Y199 was transformed with various expression vectors and grown in appropriate selective media. Co-immunoprecipitation experiments were performed essentially as described (37). Briefly, 100 µg of yeast lysate were combined with 2 µg of a monoclonal antibody recognizing the GAL4 DNA-binding domain (Clontech) and 30 µl of a 50% slurry of Protein A Sepharose beads (Pharmacia) in 1 ml of RIPA buffer containing protease inhibitors. After rocking at 4°C for 2 h, the mixtures were centrifuged at 500 g and the supernatant removed. The beads were then washed three times with RIPA buffer and resuspended in 10 µl of 2× SDS-PAGE sample dye. Subsequently, the samples were subjected to electrophoresis and immunoblotting using an anti-GAL4 activation domain antibody as described above.
RESULTS
An SV40 T antigen fragment binds an N-terminal fragment of human Top1 invitro
The N-terminus (amino acids 2-250) of human Top1 (Atop1) was expressed in bacteria as a GST fusion protein and covalently linked to glutathione-Sepharose beads for use in a secondary screen of putative Top1-binding proteins identified in a yeast two-hybrid assay (38). Yeast lysates were prepared from strains transformed with plasmids expressing fusion proteins containing various polypeptides linked to the activation domain of the GAL4 protein (AD). The lysates were incubated with Atop1-loaded beads; proteins remaining bound to the beads after several washes were detected by immunoblotting using an antibody directed against the AD. In an early experiment, we used lysates containing an AD-T antigen fusion protein containing amino acids 84-708 of SV40 T antigen as an expected negative control. The AD-T antigen fusion protein is identified as an [sim]90 Mr band upon immunoblotting with an anti-AD antibody, whereas the AD protein alone appears as a 25 Mr band (Fig. 1, lanes 1 and 3). Surprisingly, we found that the AD-T antigen fusion protein was capable of binding Atop1-loaded beads (Fig. 1, lane 5). Under these conditions, the AD protein alone did not bind to the Atop1-beads (Fig. 1, lane 2). In addition, the AD-T antigen fusion protein did not bind to beads containing GST alone (Fig. 1, lane 4). These results exclude the possibilities that the AD-T antigen-Atop1 interaction is due to either binding of AD by Atop1, or to binding of the AD-T antigen fusion protein by GST. Since both Top1 and T antigen can bind DNA, we also performed binding assays in the presence of 100 µg/ml of ethidium bromide, which impairs DNA-protein interactions (39). Binding of AD-T antigen to Atop1 was not impaired in the presence of ethidium bromide, suggesting that DNA binding is not required for the interaction between the two proteins (Fig. 1, lane 6).
Figure 2. SV40 T antigen co-immunoprecipitates with an N-terminal fragment of human topoisomerase I. Yeast were co-transformed with plasmids expressing the following proteins as indicated: the GAL4 activation domain (AD), the GAL4 binding domain (BD), a GAL4 binding domain-topoisomerase I fusion protein (BD-A1top1; containing amino acids 1-250 of Top1), or a GAL4 activation domain-T antigen fusion protein (AD-T Ag). Lysates were either directly loaded onto an SDS-PAGE gel (lanes labeled lysate), or first subjected to immunoprecipitation using an antibody recognizing BD. Immunoblotting was performed with an antibody recognizing AD. The migration of the AD-T Ag fusion protein is indicated by an arrow and the migration of the AD protein alone is indicated by an asterisk. Bands representing immunoglobulin (Ig) are also indicated. To further investigate the T antigen-Top1 interaction, we constructed a yeast expression plasmid (A1top1) encoding a fusion protein containing amino acids 1-250 of human Top1 linked to the DNA-binding domain of the GAL4 protein (BD). Yeast were co-transformed with this vector and a vector expressing either AD or the AD-T antigen fusion protein. As a control, yeast were also co-transformed with the AD-T antigen vector and a plasmid expressing BD alone. Co-immunoprecipitation studies using these co-transformants indicated that the AD protein was not detectable in anti-BD immunoprecipitates of lysates from yeast expressing AD and BD-A1top1 (Fig. 2). In contrast, AD-T antigen was co-immunoprecipitated by this antibody in lysates from yeast expressing AD-T antigen and BD-A1top1 (Fig. 2). Furthermore, AD-T antigen was not detectable in anti-BD immunoprecipitates of lysates from yeast expressing AD-T antigen and BD (Fig. 2). These data indicate that binding of the AD-T antigen fusion protein to a BD-A1top1 fusion protein is detectable by co-immunoprecipitation and that this interaction is not due to binding of BD by T antigen, or to binding of AD by Top1. 
SV40 T antigen interacts with the N-terminus of human Top1 in yeast
In order to assess whether or not the N-termini of human Top1 and T antigen are capable of interacting in intact cells, we used a modified yeast two-hybrid assay. Because of concerns about sequestration of expressed Top1 in nuclear regions that might be inaccessible to the commonly used chromosomally integrated [beta]-galactosidase reporter system (data not shown), we constructed a Saccharomyces cerevisiae strain, Y199, that contains a 2 µM-based [beta]-galactosidase reporter plasmid (27,40). Expression of BD-A1top1 alone or in combination with AD in this strain did not result in [beta]-galactosidase activity detectable by a filter-lift assay (Fig. 3). Similarly, [beta]-galactosidase activity was not detected in Y199 expressing AD-T antigen alone or in combination with BD (Fig. 3). In contrast, [beta]-galactosidase activity was detectable in colonies co-expressing AD-T antigen and BD-A1top1 (Fig. 3). As expected based upon the known interaction between T antigen and p53 (41), [beta]-galactosidase activity was also observed in colonies co-expressing AD-T antigen and BD-p53 (Fig. 3).
Figure 3. SV40 T antigen interacts with the N-terminus of human topoisomerase I in yeast. A yeast strain containing a plasmid-based [beta]-galactosidase reporter system was used to analyze topoisomerase I-binding proteins in vivo. This strain was co-transformed with plasmids expressing the indicated proteins (Fig. 2). After 5 days of growth on the appropriate selective media, [beta]-galactosidase activity was analyzed using a filter-lift assay and 5-bromo-4-chloro-3-indoyl-[beta]-d-galactopyranoside. Using this assay, colonies that express [beta]-galactosidase appear blue.
Identification of SV40 T antigen-binding regions in human Top1
We next used a series of deletion fragments of human Top1 to investigate the binding site for T antigen. Initial binding assays were performed with lysates from yeast expressing the AD-T antigen fusion protein and with Top1 peptide fragments covalently linked to Sepharose beads. The results indicated that a GST fusion protein containing Atop1 (amino acids 2-250) was capable of binding the T antigen fusion protein, whereas the GST protein alone was insufficient (Fig. 4). A GST-fusion protein containing the entire coding region of Top1 was also capable of binding T antigen (Fig. 4). Within the N-terminus of Top1, a fragment containing amino acids 1-139 was found to be necessary and sufficient for T antigen binding (Fig. 4). In addition, a fragment of Top1 lacking the N-terminus (containing amino acids 383-765) was also capable of binding T antigen under these conditions (Fig. 4). An identical binding pattern was obtained using purified T antigen rather than yeast lysates (data not shown). Taken together, these results suggest that Top1 contains two discrete sites capable of binding T antigen: one site contained within the 1-139 region and the other within the 383-765 region.
Figure 4. Analysis of T antigen-binding regions in human topoisomerase I. Sepharose beads covalently linked to either GST, full-length human topoisomerase I (FL), or the indicated amino acids of topoisomerase I were incubated with 100 µg of yeast lysate containing a GAL4 activation domain-T antigen fusion protein. After several washes, T antigen fusion protein remaining bound to the beads was identified by immunoblotting with an antibody recognizing the GAL4 activation domain. The lane labeled lysate represents 20 µg of yeast lysate loaded directly onto the gel. Since we have shown previously that the 166-210 region of Top1 is necessary and sufficient for binding nucleolin (25), we questioned whether T antigen and nucleolin could be bound simultaneously on the N-terminus of Top1. In these experiments we used purified T antigen and GST-nucleolin, which migrate with apparent molecular weights of [sim]80 and 120 Mr, respectively (Fig. 5, lanes 1 and 2). Similar to the AD-T antigen fusion protein, purified T antigen is capable of binding Atop1 and the Top1 holoenzyme in vitro (Fig. 5, lanes 4 and 9). The lack of detectable silver-stained peptides co-binding to Atop1 with T antigen suggests that stoichiometric quantities of other proteins are not required for the interaction between T antigen and the N-terminus of Top1 (Fig. 5, lane 4). In experiments involving mixtures of T antigen and GST-nucleolin, a quantity of T antigen was added that was sufficient to almost saturate binding to the Atop1 beads (data not shown). Under these conditions, while binding of T antigen to Atop1 is maintained in the presence of an equimolar quantity of GST-nucleolin (Fig. 5, lane 6), T antigen binding is diminished in the presence of excess GST-nucleolin (Fig. 5, lanes 7 and 8). These data suggest that although T antigen and nucleolin may be bound to Top1 concurrently (consistent with the identification of distinct binding sites), excess nucleolin may displace T antigen from Top1. Further studies will be necessary to elucidate the mechanisms underlying the latter phenomenon. Figure 5. Concurrent binding of nucleolin and T antigen to the N-terminus of human topoisomerase I. Purified T antigen and GST-nucleolin were either directly loaded onto a 7.5% polyacrylamide gel (lanes 1 and 2), or were incubated with beads covalently linked to either GST (labeled a), a peptide containing amino acids 2-250 of topoisomerase I fused to GST (labeled b) or full-length human topoisomerase I fused to GST (labeled c). Binding of T antigen to beads containing the 2-250 N-terminal Top1 peptide was assessed at saturating levels of T antigen in the absence (lane 4) or presence (lanes 5-8) of GST-nucleolin. GST-nucleolin was added at molar ratios of 0.2, 1, 2 or 5 relative to T antigen (lanes 5-8, respectively). After several washes, proteins remaining bound to the beads were identified by SDS-PAGE and silver staining. The first two lanes represent 1.4 µg of GST-nucleolin and 1 µg of T antigen, respectively.
DISCUSSION
In previous work we identified an interaction between the N-terminus of Top1 and nucleolin (25). Subsequently, using a yeast two-hybrid assay we have identified other proteins capable of binding the 1-250 region of Top1 (data not shown). In the course of this work we detected an interaction between this region of Top1 and a fragment of the SV40 T antigen containing amino acids 84-708. This interaction was also detectable in co-immunoprecipitation studies. Furthermore, T antigen-Atop1 binding was evident in yeast using a two-hybrid assay. Additional data suggest that binding of T antigen to the N-terminus of Top1 does not involve intermediary DNA or other proteins. Our data also imply the existence of two binding sites on Top1 for T antigen, one involving amino acids 1-139, the other contained within the 383-765 region of the protein. T antigen is required for SV40 DNA replication and is known to be a DNA and RNA helicase (42,43). Interestingly, although nucleolin has been reported to be a DNA and RNA helicase (26), there is little sequence homology shared by T antigen and this protein and the binding site for nucleolin in the N-terminus of Top1 (amino acids 166-210) is different from that of T antigen. Indeed, we are able to detect concurrent binding of T antigen and nucleolin to the N-terminus of Top1. These findings suggest that one role of the N-terminus of Top1 is to bind diverse classes of helicases and that this binding is conferred by distinct regions. This putative role for the N-terminus of Top1 is consistent with the finding that deletion of this region has little effect on topoisomerase activity (25,44,45). Although Top1 has been found to interact with non-helicase proteins such as TBP (13), p53 (46) and the SR protein SF2/ASF (15), it is not yet known whether or not these interactions involve the N-terminus of Top1.
Prior work supports a functional interrelation between T antigen and Top1. In vitro studies of SV40 DNA replication indicate that either topoisomerase I or II is required for this process (47). This requirement is explained by a model whereby a DNA helix tracking complex such as a replication fork generates positive supercoiling that must be dealt with by a topoisomerase (47,48). While this model invokes a functional inter-relationship between a helicase and a topoisomerase, it does not necessitate a physical association between the two proteins. Direct helicase-topoisomerase interactions may have evolved to ensure that positive supercoiling generated in front of a helicase-polymerase tracking complex is resolved in an efficient manner (Fig. 6). Notably, the linkage of a helicase and a topoisomerase in this context may result in a eukaryotic gyrase, since the complex would be predicted to convert a relaxed closed circular DNA into a negatively supercoiled form. A similar concept has been proposed recently by Duguet (49) and by Simmons et al., who have also identified a physical association between T antigen and Top1 (50). The data provided by Simmons et al. support a functional requirement for helicase-topoisomerase binding, since T antigen mutants that retain helicase activity but exhibit diminished affinity for Top1 are defective in plasmid unwinding activity (50,51). A physical association between T antigen and Top1 may have additional relevance to the SV40 virus, since it would increase the likelihood that cellular Top1 would be available for viral replication.
Figure 6. Model for the role of a helicase-topoisomerase interaction in DNA replication. The potential problem of local overwinding in the absence of a direct helicase-topoisomerase interaction is illustrated. The finding that helicases such as T antigen and nucleolin interact with the N-terminus of Top1 is consistent with several recent reports of interactions between helicases and other topoisomerases. The yeast protein Sgs1, which is a member of the RecQ family of helicases that also contains the proteins that are defective in Bloom's and Werner's syndromes (52,53), has been shown to bind yeast topoisomerase II and III (8,54). Similar to the Bloom's protein, Sgs1 is involved in maintenance of genomic stability (8,52). Interestingly, with regard to Sgs1, the genomic instability phenotype is not dependent on the helicase function of the protein (55), implicating another region of Sgs1 in this phenotype. Knowledge of the domains involved in helicase-topoisomerase binding should allow determination of whether or not loss of topoisomerase binding is involved in the phenotypes associated with defects in the Sgs1, Bloom's and Werner's proteins.
ACKNOWLEDGEMENTS
The authors would like to thank Dr Steven J.Brill for providing purified T antigen and Drs Leroy F.Liu, Marc R.Gartenberg and Nancy C.Walworth for helpful comments regarding this work. This investigation was supported by United States Public Health Service Grant CA70981, awarded by the National Cancer Institute.
REFERENCES
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