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• W2014825046 abstract "Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluorescein-conjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time- and energy-dependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin α, importin β, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import. Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluorescein-conjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time- and energy-dependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin α, importin β, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import. The nuclear envelope presents an effective barrier between the nuclear and cytoplasmic compartments of the cell. Although it is impermeant to large non-nuclear molecules, a multitude of macromolecules must enter and exit the nucleus across this envelope every second in order for the cell to live. All macromolecular exchange between the nucleus and the cytoplasm studied to date occurs through the nuclear pore complex (NPC), 1The abbreviations used are: NPC, nuclear pore complex; NLS, nuclear localization signal; HIV, human immunodeficiency virus; GFP, green fluorescent protein; PNA, peptide nucleic acid; Fl, fluorescein; Rh, rhodamine; BSA, bovine serum albumin; BSA-NLS, synthetic NLS peptide-conjugated bovine serum albumin; WGA, wheat germ agglutinin; pDNA, plasmid DNA; bp, base pair(s); kb, kilobase(s). is signal-dependent, and utilizes a series of receptor proteins (for a review see Ref. 1Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1011) Google Scholar). In the case of proteins destined for the nucleus, the nuclear localization signal (NLS) interacts with one of a growing number of importin family members to target the complex to the NPC. In the classical case of NLS-containing proteins, the protein binds to importin α, the NLS “receptor,” which in turn interacts with importin β. Once at the NPC, the complex interacts with the small GTP-binding protein RAN in its GDP-bound state and its accessory factor NTF2 while being translocated across the NPC. After translocation into the nucleus, the complex disassembles because of the conversion of RAN-GDP to RAN-GTP by exchange or replacement and the importins return to the cytoplasm (2Melchior F. Gerace L. Trends Cell Biol. 1998; 8: 175-179Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Similar scenarios of receptor proteins interacting with signals to mediate translocation across the nuclear envelope also occur for the nuclear export of proteins (e.g. exportin and the nuclear export signal) and viral mRNAs (Crm1p, HIV Rev, and the Rev response element), and the nuclear import of small nuclear RNAs that contain both protein-encoded NLSs and the trimethylguanosine cap as import signals (1Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1011) Google Scholar, 3Moroianu J. J. Cell. Biochem. 1998; 70: 231-239Crossref PubMed Scopus (60) Google Scholar). We have recently shown that the nuclear import of plasmid DNA (pDNA) also uses a signal-mediated pathway to enter the nucleus. Using a microinjection approach, we demonstrated that pDNA can enter the nucleus in the absence of cell division by a process that is consistent with transport through the NPC (4Dean D.A. Exp. Cell Res. 1997; 230: 293-302Crossref PubMed Scopus (234) Google Scholar). Such nuclear import has been detected in all mammalian cells tested to date, including those of mouse, rat, monkey, human, and chicken, as well as those from all cell types, including smooth and striated muscle, fibroblasts, endothelial cells, and epithelial cells. Furthermore, this import is sequence-specific: plasmids containing as little as 80 bp of the SV40 enhancer/early promoter region are targeted to the nucleus in the absence of cell division, whereas plasmids lacking this sequence remain cytoplasmic (4Dean D.A. Exp. Cell Res. 1997; 230: 293-302Crossref PubMed Scopus (234) Google Scholar). 2D. A. Dean, B. S. Dean, S. Muller, and L. C. Smith, submitted for publication. This sequence specificity appears unique to SV40, because several other strong viral promoter and enhancer sequences, including the immediate early promoter/enhancer of cytomegalovirus and the long terminal repeat of Rous sarcoma virus, fail to promote nuclear import.2Based on the sequences required for plasmid nuclear import, we have proposed a working model in which pDNA import is mediated by newly synthesized transcription factors and other sequence-specific DNA-binding proteins that bind to the DNA in the cytoplasm, thus attaching NLSs to the DNA for recognition and transport by the NLS-dependent machinery. In order to further study the mechanisms of pDNA nuclear import, an in vitro system was required. To this end, we have developed an approach to label plasmid DNA that maintains its biological activity and have adapted the digitonin-permeabilized cell assay to study plasmid nuclear localization. The 4.2-kb plasmid pUSAG3 contains a 60-bp sequence from the human G gamma globin promoter (−315 to −255) inserted into the SmaI site of the GFP promoter reporter vector pEGFP-1 (CLONTECH Laboratories, Palo Alto, CA) in the same orientation as the GFP gene (see Fig. 1). To create pUSAG3ΔSV40 that lacks the SV40 nuclear targeting sequence, a 913-bp fragment containing the SV40 origin and early promoter/enhancer region was removed from pUSAG3 by digestion with StuI and SspI and religation. pUSAG9 is a 14.4-kb plasmid containing the human globin locus control region and the A-γ globin and β globin genes (nucleotides 38084–43975 and 60409–65475, EMBL ID: HSHBB4R1) inserted into the SmaI site of pEGFP-1. pUSAG9 also contains the 60-bp PNA-binding site found in pUSAG3. Rhodamine-labeled pGenegrip blank vector and Rhodamine-labeled pGenegrip GFP vector were obtained from Gene Therapy Systems (San Diego, CA). A fluorescein-labeled PNA clamp (NH2-fluorescein-OTTTTCTTCTCOOOJTJTTJTTTT-COOH; Fl-PNA) was synthesized by Perseptive Biosystems (Framingham, MA) to bind to the target GAGAAGAAA present in the 60-bp G γ globin promoter fragment (see Fig. 1). Fl-PNA (3 μm) was reacted with the purified plasmids at a 10:1 molar ratio in TE buffer (10 mm Tris, pH 8, 1 mm EDTA) for 3 h at 37 °C. The reactions were diluted into 2 ml of TE buffer and concentrated to 50 μl in a Centricon-30 microconcentration device, diluted again to 2 ml, and reconcentrated to a final concentration of between 0.1 and 0.2 mg/ml plasmid. Approximately 30 μg of pDNA was labeled in a typical experiment. Labeled DNAs were stored at −80 °C. The pGenegrip vectors were obtained as labeled DNAs from the manufacturer. HeLa cells were grown on coverslips in minimum essential medium (Life Technologies, Inc.) containing 10% fetal bovine serum and antibiotics in a humidified incubator at 37 °C with 5% CO2. Purified PNA-labeled pDNA was suspended in phosphate-buffered saline at a concentration of 0.1 mg/ml and microinjected into the cytoplasm of cells grown on etched coverslips as described (4Dean D.A. Exp. Cell Res. 1997; 230: 293-302Crossref PubMed Scopus (234) Google Scholar). With a microinjection volume of approximately 3 × 10−10 ml/cell,2 about 2,000 plasmids were delivered per cell. The cells were incubated for various times, rinsed in phosphate-buffered saline, fixed for 15 min at 4 °C in 3% paraformaldehyde in phosphate-buffered saline, and mounted with 4,6-diamidino-2-phenylindole/diazobicyclo[2.2.2]octane. The human genes for importin β and RAN were polymerase chain reaction-amplified from reverse-transcribed HeLa cell RNA using oligonucleotide primers containing unique restrictions sites at the ends and cloned into the corresponding restriction sites of ptrcHisB (Invitrogen, San Diego, CA). A plasmid expressing the yeast importin α homologue, hSRP1α, was obtained from K. Weis (EMBL, Heidelberg, Germany) (5Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (309) Google Scholar). The plasmids were transformed intoEscherichia coli BL21(DE3) and expressed and purified by nickel affinity chromatography as described for the appropriate protein (5Weis K. Mattaj I.W. Lamond A.I. Science. 1995; 268: 1049-1053Crossref PubMed Scopus (309) Google Scholar, 6Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2008-2011Crossref PubMed Scopus (251) Google Scholar). Permeabilized cell assays were performed as described previously using HeLa cells grown on glass coverslips (7Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (771) Google Scholar). Briefly, cells were grown to 60% confluency, washed with wash buffer (20 mm HEPES, pH 7.3, 110 mmpotassium acetate, 5 mm sodium acetate, 2 mmMgCl2, 1 mm EGTA, 2 mmdithiothreitol), and permeabilized with 40 μg/ml digitonin in wash buffer for 6–8 min on ice. The cells were rinsed for 5–10 min with several changes of cold wash buffer, and the excess buffer was removed before the assays were initiated. Reactions were carried out in a transport buffer consisting of wash buffer containing 1 mg/ml bovine serum albumin, 1 μg/ml aprotinin, leupeptin, and pepstatin, 1 mm GTP, 2 mm ATP, 10 mmphosphocreatine, and 20 units/ml of creatine phosphokinase. Rhodamine- or fluorescein-labeled NLS peptide-conjugated BSA (Fl- or Rh-BSA-NLS) were included at 25 μg/ml and fluorescein-labeled PNA plasmids were present at 10 μg/ml. Where indicated, cytoplasmic extracts prepared from HeLa cells (7Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (771) Google Scholar) were added to between 5 and 7 mg/ml, HeLa cell nuclear extracts (in vitro transcription grade, Promega, Madison, WI) were added to 0.25 mg/ml, and affinity purified, recombinant his-tagged RAN, importin α, and importin β were used at 0.5 mg/ml each. The lectins wheat germ agglutinin (WGA) and concanavalin A were added to 0.1 mg/ml each. In reactions lacking ATP and GTP, nucleotide triphosphates, phosphocreatine, and creatine-phosphokinase were omitted from the transport buffer and ATP-depleted extracts were prepared by incubating the extracts with 10 units/ml apyrase for 30 min before addition to the cells. Other competitors were added as indicated in the text. The cells were incubated in a humidified chamber at 37 °C for 4 h, unless otherwise indicated. The reactions were terminated by washing the cells in wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. The cells were mounted with 4,6-diamidino-2-phenylindole/diazobicyclo[2.2.2]octane, and 0.5-μm sections were viewed by confocal microscopy using an ACAS 570 laser-scanning confocal microscope. PNA-labeled and unlabeled plasmids were incubated in transport buffer containing HeLa cytoplasmic and nuclear extracts (5 and 0.25 mg/ml, respectively) for 4 h at 37 °C. At the end of the reaction, the DNA was extracted with phenol:chloroform and chloroform alone. 10 μg of tRNA were added, and the DNA was precipitated with ethanol overnight at −20 °C. The DNA was resuspended in 10 μl of TE buffer and separated on a 1% agarose gel, transferred to nylon, and immobilized by UV irradiation as described (8Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1994Google Scholar). The Blot was prehybridized at 42 °C for 4 h in 50% formamide containing 5× SSC, 5× Denhardt's solution, and 2 mg/ml sheared salmon sperm DNA and hybridized with 1 × 106cpm/ml of nick translated pUSAG3 and 1 × 106 cpm/ml of nick translated pGenegrip Blank for 18 h at 37 °C. The blot was washed at room temperature for 15 min each in 2× SSC/0.1% SDS, 0.5× SSC/0.1% SDS, 0.5× SSC/0.1% SDS, and for 30 min at 37 °C in 0.1× SSC/1% SDS, dried, and exposed to film. Although we have begun to make progress elucidating the mechanisms of nuclear import of plasmid DNA using microinjection and in situ hybridization, this method has its limitations because of the fact that it detects DNA in fixed samples only. A better substrate with which to follow the transport reactions in real time would be fluorescently labeled pDNA. In order to produce such substrate, we tried a variety of labeling techniques including incorporation of fluorescent nucleotide analogues (e.g. fluorescein-dUTP) by polymerase chain reaction or nick translation followed by ligation to form intact plasmid, covalent and noncovalent high affinity intercalating dyes (e.g. ethidium bromide monoazide and TOTO-1), and reaction of supercoiled plasmid with photoactivatable fluorophore conjugates. Although all of the methods produced fluorescently labeled DNA, two problems limited the use of these labeled DNAs as substrates for import reactions. First, several of the techniques produced either linear DNA or low yields of circularized plasmid, making their use impractical. More importantly, all of the labeled DNAs became inactive in both transcription of reporter genes or migration of the DNA to the nucleus in microinjected cells (not shown). We were successful, however, when we used a fluorescently labeled peptide nucleic acid (Fl-PNA) clamp; the resulting plasmids were transcriptionally active and able to localize to the nucleus as did unlabeled native plasmid. 3Wang, G., Xu, X., Pace, B., Dean, D. A., Glazer, P. M., Chan, P., Goodman, S. R., and Shoklenko, I. (1999) Nucleic Acids Res. 27, 2806–2813. The PNA we used to label plasmids bound to a 10-nucleotide sequence (GAGAAGAAAA) within the G γ globin promoter. The target site was cloned upstream of the GFP gene, well removed from the SV40 nuclear targeting sequence, which was downstream of the GFP gene, 1.6 kb away (Fig. 1). One half of the PNA invaded the target site and hybridized to its complementary sequence through standard Watson-Crick base pairs, and the other half of the PNA folded back onto the PNA:DNA double helix to form a triplex structure using Hoogstein base pairs (9Egholm M. Buchardt O. Christensen L. Behrens C. Freier S.M. Driver D.A. Berg R.H. Kim S.K. Norden B. Nielsen P.E. Nature. 1993; 365: 566-568Crossref PubMed Scopus (1786) Google Scholar). Fluorescein was bound to the amino terminus of the PNA and did not interfere with binding of the PNA to the target. The resulting triplex structure is highly stable (10Nielsen P.E. Egholm M. Buchardt O. Gene (Amst .). 1994; 149: 139-145Crossref PubMed Scopus (165) Google Scholar), and because the backbone of the PNA utilizes peptide bonds, it is resistant to nuclease digestion and even incubation in serum (11Demidov V.V. Potaman V.N. Frank-Kamenetskii M.D. Egholm M. Buchardt O. Sonnichsen S.H. Nielsen P.E. Biochem. Pharmacol. 1994; 48: 1310-1313Crossref PubMed Scopus (621) Google Scholar). In our hands, the PNA could not be dissociated from the bound state when incubated with a 1000-fold molar excess of target sequence at 37 °C for 8 h as followed by gel shift assays (not shown). When microinjected into the cytoplasm of TC7 cells, native pDNA localizes to the nucleus within 6–8 h as detected by in situ hybridization (4Dean D.A. Exp. Cell Res. 1997; 230: 293-302Crossref PubMed Scopus (234) Google Scholar). When Fl-PNA-labeled pUSAG3 was similarly microinjected into the cytoplasm, it localized to the nucleus within 6 h (Fig. 2). Further, the subnuclear distribution of the labeled pDNA and its absence in the nucleoli was very similar to that seen with native pDNA and in situ hybridization (4Dean D.A. Exp. Cell Res. 1997; 230: 293-302Crossref PubMed Scopus (234) Google Scholar). Thus, because the Fl-PNA/pDNA maintained the same biological properties as unmodified plasmid, we used it as a substrate in permeabilized cell assays to dissect the transport mechanism. Digitonin-permeabilized HeLa cells have been used effectively to reconstitute nuclear import and export reactions of proteins, small nuclear RNA, and small linear DNA (7Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (771) Google Scholar, 12Marshallsay C. Lührmann R. EMBO J. 1994; 13: 222-231Crossref PubMed Scopus (62) Google Scholar, 13Hagstrom J.E. Ludtke J.J. Bassik M.C. Sebestyén M.G. Adam S.A. Wolff J.A. J. Cell Sci. 1997; 110: 2323-2331PubMed Google Scholar, 14Holaska J.M. Paschal B.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14739-14744Crossref PubMed Scopus (38) Google Scholar, 15Love D.C. Sweitzer T.D. Hanover J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10608-10613Crossref PubMed Scopus (87) Google Scholar). We also have used this system to study the requirements for fluorescently labeled pDNA nuclear entry. HeLa cells were permeabilized with digitonin and washed to remove cytoplasmic components as described previously (7Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (771) Google Scholar). Because all signal-mediated nuclear import reported to date has been shown to require cytosolic proteins (e.g.importins, exportins, etc.), we reasoned that signal-mediated pDNA nuclear import would behave similarly. Thus, we added cytoplasmic extracts prepared from HeLa cells along with an ATP-regenerating system, protease inhibitors, buffer, salts, and GTP to the assays. As a positive control for nuclear import, we also followed the nuclear accumulation of rhodamine-labeled BSA conjugated to a synthetic NLS peptide (Rh-BSA-NLS) to ensure that all the components were active (not shown). As seen in Fig. 3, significant Fl-PNA/pDNA import is observed within 90 min and is maximal by 4 h. In contrast, Rh-BSA-NLS was maximally imported into the nuclei by 30 min (not shown). That the extract was still fully functional and that the nuclei were not leaky at these late times were confirmed by demonstrating that protein nuclear import occurred in an NLS-dependent manner when the substrate was added 4–8 h after the cells had been permeabilized and reacted with cytoplasmic extracts (not shown). Thus, neither the cells nor the extracts lost functional activity or selectivity over this time course. Because maximal nuclear localization was observed by 4 h, this time point was used in the remainder of the experiments. To determine whether cytoplasmic extracts were indeed required for the nuclear import of pDNA, reactions were performed with various combinations of cellular extracts (Fig. 4). Our working model for signal-mediated pDNA nuclear import postulates that sequence specificity is mediated by transcription factors and DNA-binding proteins. These proteins bind to sites within the SV40 DNA nuclear targeting sequence while the DNA and the proteins are in the cytoplasm, and the NLSs present on the transcription factors enable the pDNA-protein complex to utilize the NLS-dependent machinery for nuclear import. This model predicts that cytoplasmic extracts containing both DNA-binding proteins and the NLS-dependent import machinery are necessary for nuclear import of pDNA. In support of this model, no nuclear import was observed in the absence of cytoplasmic extract of either a 4.2-kb plasmid, pUSAG3, or a 14.4-kb plasmid, pUSAG9, both of which contain the PNA-binding site and the SV40 DNA nuclear targeting sequence (Fig. 4, A and E). In contrast, when cytoplasmic extract was provided, significant nuclear import of both plasmids was detected at 4 h (Fig. 4, C and G). Interestingly, many nuclei showed distinct rim staining of the larger plasmid (Fig. 4G). This suggests either that import was not complete at 4 h and larger plasmids take longer to enter the nucleus or that one or more factors are limiting for efficient nuclear import of the larger plasmid. Because our model predicts that transcription factors and other DNA-binding proteins mediate nuclear pDNA import, nuclear extracts were tested for their ability to support or enhance nuclear import of the plasmids. Although the addition of nuclear extract alone did not support nuclear localization of the 14.4-kb plasmid (Fig. 4F), it did allow a low level of import of the smaller plasmid (Fig. 4B). Because all components of the NLS-dependent import machinery can be found in the nucleus at levels much less than those in the cytoplasm, this is not a completely surprising result. When both nuclear and cytoplasmic extracts were provided to the cells, import of both plasmids was more robust (Fig. 4, D and H). Further, no rim staining was observed for either of the plasmids. Signal-mediated nuclear import and export of proteins and RNAs has been shown to be energy-dependent (7Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (771) Google Scholar, 12Marshallsay C. Lührmann R. EMBO J. 1994; 13: 222-231Crossref PubMed Scopus (62) Google Scholar, 14Holaska J.M. Paschal B.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14739-14744Crossref PubMed Scopus (38) Google Scholar, 15Love D.C. Sweitzer T.D. Hanover J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10608-10613Crossref PubMed Scopus (87) Google Scholar). Previous experiments from our laboratory using a microinjection approach have demonstrated that this is also the case for plasmid nuclear import (4Dean D.A. Exp. Cell Res. 1997; 230: 293-302Crossref PubMed Scopus (234) Google Scholar). This energy dependence was confirmed in the permeabilized cell system (Fig. 5). When GTP and the ATP-regenerating system were omitted from the assays and endogenous stores of nucleotide triphosphates were depleted from the cytoplasmic and nuclear extracts by pretreatment with apyrase, no nuclear localization of pDNA was detected with any combination of extracts (Fig. 5). Similarly, these treatments also prevented nuclear accumulation of Rh-BSA-NLS (data not shown), confirming that ATP levels were sufficiently low to block NLS-mediated nuclear import. Addition of the lectin WGA that binds to N-acetylglucosamine residues present on a class of NPC proteins also inhibited nuclear accumulation of pDNA in permeabilized cells (Fig. 6). When added to cells, WGA completely inhibited the nuclear import of Fl-PNA/pUSAG3 (Fig. 6C) compared with the control reaction lacking added lectin (Fig. 6A), whereas addition of the lectin concanavalin A that has been shown not to alter transport through the NPC had no effect on pDNA import (Fig. 6B). Based on the above experiments, it appeared that the need for cytoplasmic extracts for pDNA nuclear import was to supply components of the NLS-dependent transport machinery. Thus, we expressed and affinity purified histidine-tagged fusion proteins corresponding to importin α, importin β, and RAN. The proteins were judged to be >95% pure based on Coomassie Blue-stained polyacrylamide gels (data not shown). These were then added with either Rh-BSA-NLS or Fl-PNA/pUSAG3 to permeabilized cells, in the presence or absence of nuclear extract prepared from HeLa cells to provide the appropriate NLS-containing DNA-binding proteins (Fig. 7). As seen previously, in the absence of cytoplasmic extract or the importins and RAN, no nuclear import of either pDNA or Rh-BSA-NLS was observed (Fig. 7, A and E). Similarly, little or no nuclear import of either substrate was seen in the presence of nuclear extract alone (Fig. 7,B and F). Although the addition of importin α, importin β, and RAN was sufficient to drive the nuclear localization of the NLS-containing reporter protein (Fig. 7G), they were not sufficient to support import of pDNA (Fig. 7C). This suggested either that plasmid DNA does not use the importin α/β pathway for its nuclear import or that additional factors are required to allow the importins to bind to the DNA. Because neither importin α nor β bind to DNA, for them to mediate nuclear import of pDNA, an intermediary “adapter” protein would be required that binds to DNA and contains an NLS for recognition by the importins. Thus, when a nuclear extract was provided in addition to the importins and RAN, significant nuclear localization of Fl-PNA/pUSAG3 was observed (Fig. 7D). The presence of the nuclear extract had little effect on the import of Rh-BSA-NLS (Fig. 7H). To ensure that all three components of the nuclear import machinery (i.e. importin α, importin β, and RAN) were indeed required for pDNA nuclear entry, import reactions were performed in which each of the factors was left out separately (Fig. 8). As was seen in Fig. 7, significant nuclear import of both pDNA and Rh-BSA-NLS was observed in the presence of all three components and nuclear extract (Fig. 8, A and E). However, when the import reactions were performed with nuclear extract and importin α and RAN (Fig. 8, B and F), importin β and RAN (Fig. 8, C and G), or importin α and importin β (Fig. 8, Dand H), nuclear import of both substrates was eliminated. The nuclear rim staining of Rh-BSA-NLS observed in the presence of the importins alone (Fig. 8H) was expected based on previous reports (16Moore M.S. Blobel G. Cell. 1992; 69: 939-950Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 17Zelphati O. Liang X. Hobart P. Felgner P.L. Hum. Gene Ther. 1999; 10: 15-24Crossref PubMed Scopus (111) Google Scholar) and confirms the fidelity of the recombinant proteins and the experimental system. Thus, similar to “classical” protein nuclear import, both the importins and RAN are required for plasmid nuclear entry. Competition experiments suggested that the nuclear import of Fl-PNA/pDNA was signal-mediated. When permeabilized cells were incubated with Fl-PNA/pUSAG3 in the presence of ATP and cytoplasmic and nuclear extracts, the plasmid localized to the nuclei of cells (Fig. 9A). Addition of a 1000-fold molar excess of BSA-NLS had no effect on the nuclear localization of the plasmid (Fig. 9B). This is not entirely surprising, because the small NLS-containing protein is imported into the nuclei within minutes of addition and thus is not a true competitor for the NLS-dependent machinery, which can recycle back to the cytoplasm for import of pDNA. Striking inhibition of pDNA nuclear import was seen when a 100-fold molar excess of SV40 DNA was added to the reaction (Fig. 9D), whereas a similar excess of pUC19 had no effect on pDNA import (Fig. 9C). Because both pUSAG3 and SV40 DNA share the 360-bp SV40 promoter/enhancer region that is necessary in intact cells for nuclear targeting of pDNA, it appeared likely that the permeabilized cell system faithfully reproduced this sequence specificity. To test this directly, a 913-bp fragment containing the 360-bp SV40 DNA nuclear targeting sequence was removed from pUSAG3 to create pUSAG3ΔSV40. This plasmid was labeled with Fl-PNA and used in the permeabilized cell assay in parallel to the parent Fl-PNA/pUSAG3 (Fig. 10). As expected, neither plasmid was able to localize to the nucleus in the absence of cytoplasmic extract (Fig. 10, A and C). The plasmid lacking the SV40 sequence was also unable to localize to the nuclei in the presence of cytoplasmic extract, although faint rim staining was detected (Fig. 10D), whereas the parent pUSAG3 was imported efficiently (Fig. 10B). These experiments confirm that pDNA nuclear import in permeabilized cells faithfully represents that seen in intact cells and that nuclear uptake of plasmid DNA is sequence-specific.Figure 10Sequence specificity of pDNA nuclear import in permeabilized cells. Permeabilized HeLa cells were incubated for 4 h at 37 °C in transport buffer containing BSA alone (A and C) or HeLa cytoplasmic extract at 5 mg/ml (B and D). The substrates used were either Fl-PNA/pUSAG3 (A and B) or Fl-PNA/pUSAG3ΔSV40 (C and D), both present at 10 μg/ml.View Large Image Figure ViewerDownload Hi-res image Download (P" @default.
• W2014825046 created "2016-06-24" @default.
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• W2014825046 date "1999-07-01" @default.
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• W2014825046 title "Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences" @default.
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