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Distinct Effects of Topoisomerase I and RNA Polymerase I Inhibitors Suggest a Dual Mechanism of Nucleolar/Nucleoplasmic Partitioning of Topoisomerase I

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m400498200

ISSN

1083-351X

Autores

Morten O. Christensen, René M. Krokowski, Hans Barthelmes, Robert Hock, Fritz Boege, Christian Mielke,

Tópico(s)

Neutropenia and Cancer Infections

Resumo

Topoisomerase I is mostly nucleolar, because it plays a preeminent role in ribosomal DNA (rDNA) transcription. It is cleared from nucleoli following exposure to drugs stabilizing covalent DNA intermediates of the enzyme (e.g. camptothecin) or inhibiting RNA polymerases (e.g. actinomycin D), an effect summarily attributed to blockade of rDNA transcription. Here we show that two distinct mechanisms are at work: (i) Both drugs induce inactivation and segregation of the rRNA transcription machinery. With actinomycin D this leads to a co-migration of RNA-polymerase I and topoisomerase I to the nucleolar perimeter. The process has a slow onset (>20 min), is independent of topoisomerase I activity, but requires the N-terminal domain of the enzyme to colocalize with RNA polymerase I. (ii) Camptothecin induces, in addition, immobilization of active topoisomerase I on genomic DNA resulting in rapid nucleolar clearance and spreading of the enzyme to the entire nucleoplasm. This effect is independent of the state of rRNA transcription, involves segregation of topoisomerase I from RNA polymerase I, has a rapid onset ( 20 min), is independent of topoisomerase I activity, but requires the N-terminal domain of the enzyme to colocalize with RNA polymerase I. (ii) Camptothecin induces, in addition, immobilization of active topoisomerase I on genomic DNA resulting in rapid nucleolar clearance and spreading of the enzyme to the entire nucleoplasm. This effect is independent of the state of rRNA transcription, involves segregation of topoisomerase I from RNA polymerase I, has a rapid onset (<1 min), and requires catalytic activity but neither the N-terminal domain of topoisomerase I nor its major sumoylation site. Thus, nucleolar/nucleoplasmic partitioning of topoisomerase I is regulated by interactions with RNA polymerase I and DNA but not by sumoylation. The mechanism that distributes DNA-topoisomerase I (topo I) 1The abbreviations used are: topo I, topoisomerase I; ActD, actinomycin D; CPT, camptothecin; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; rDNA, ribosomal DNA; SUMO, small ubiquitin-like modifier; GFP, green fluorescent protein; PBS, phosphate-buffered saline; BSA, bovine serum albumin. between nucleoli and nucleoplasm has been a controversial issue over a decade. Topoisomerase I catalyzes topological changes in the DNA by cutting one strand of the double helix and allowing rotation of the other (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2192) Google Scholar). This mechanism releases torsion stress in DNA double helices that is created by e.g. RNA synthesis. Thus, topo I is a crucial cofactor for the transcription of nucleoplasmic genes (2Stewart A.F. Schutz G. Cell. 1987; 50: 1109-1117Abstract Full Text PDF PubMed Scopus (122) Google Scholar) and rDNA (3Muller M.T. Pfund W.P. Mehta V.B. Trask D.K. EMBO J. 1985; 4: 1237-1243Crossref PubMed Scopus (167) Google Scholar, 4Zhang H. Wang J.C. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1060-1064Crossref PubMed Scopus (262) Google Scholar). The latter task seems to dominate, because topo I is concentrated in the nucleoli, and, of the three nucleolar components, it is preferentially found in the fibrillar centers, a restriction in localization that is lost when the N-terminal domain of the enzyme or parts of it are lacking (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Topoisomerase I is thus placed in the vicinity of rDNA, RNA polymerase I, and rDNA transcription, which are also restricted to the fibrillar centers of the nucleolus (6Cheutin T. O'Donohue M.F. Beorchia A. Vandelaer M. Kaplan H. Defever B. Ploton D. Thiry M. J. Cell Sci. 2002; 115: 3297-3307PubMed Google Scholar). Nucleolar positioning of topo I is highly susceptible to exogenous perturbation. The enzyme is lost from fibrillar centers, when cells are cultured at an inappropriate temperature (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). It is also lost from nucleoli in response to camptothecin and related compounds that inhibit the religation step of the DNA cleavage-religation mechanism and thus stabilize the enzyme in covalent DNA intermediates (7Danks M.K. Garrett K.E. Marion R.C. Whipple D.O. Cancer Res. 1996; 56: 1664-1673PubMed Google Scholar, 8Buckwalter C.A. Lin A.H. Tanizawa A. Pommier Y.G. Cheng Y.C. Kaufmann S.H. Cancer Res. 1996; 56: 1674-1681PubMed Google Scholar). Because such compounds also inhibit RNA synthesis (9Kaufmann S.H. Cancer Res. 1991; 51: 1129-1136PubMed Google Scholar) and because direct inhibitors of RNA polymerase I such as actinomycin D (10Perry R.P. Exp. Cell Res. 1963; 29: 400-406Crossref Scopus (198) Google Scholar) also remove topo I from the nucleoli, it has been proposed that the same cellular mechanism (i.e. loss of rRNA synthesis) is responsible for nucleolar clearance of topo I in response to both types of inhibitors (8Buckwalter C.A. Lin A.H. Tanizawa A. Pommier Y.G. Cheng Y.C. Kaufmann S.H. Cancer Res. 1996; 56: 1674-1681PubMed Google Scholar, 11Mao Y. Mehl I.R. Muller M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1235-1240Crossref PubMed Scopus (40) Google Scholar). This implies ongoing rDNA transcription to be crucial for the positioning of the enzyme in the nucleolus. Recently, we have put forward an alternative hypothesis formed on the basis of our finding that topo I is very mobile in a living cell. It roams the entire nuclear space and exchanges rapidly between nuclear compartments. Camptothecin reduces the mobility of topo I by more than 10-fold. Moreover, it acts preferentially on topo I in the nucleoplasm, where the enzyme is normally more mobile than in the nucleoli. Thus, partitioning of topo I between nucleoli and nucleoplasm seems in general governed by mobility gradients within the cell nucleus, with nucleolar accumulation reflecting the enzyme's lesser mobility in the nucleoli, and relocation to the nucleoplasm in response to camptothecin reflecting attenuation of the enzyme at nucleoplasmic sites, where it is actively processing genomic DNA (12Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, this simple explanation was disputed, because camptothecin also stimulates modification of topo I with small ubiquitin-like modifiers (SUMO) (13Mao Y. Sun M. Desai S.D. Liu L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4046-4051Crossref PubMed Scopus (181) Google Scholar), and mutational silencing of the major target site of topo I for this modification (K103R,K117R,K153R) enhances nucleolar accumulation of the enzyme and abolishes its nucleolar clearance in response to camptothecin (14Rallabhandi P. Hashimoto K. Mo Y.Y. Beck W.T. Moitra P.K. D'Arpa P. J. Biol. Chem. 2002; 277: 40020-40026Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This has been interpreted as an indication of an active and directed transport of topo I between nucleoli and nucleoplasm that is triggered by the attachment of SUMO to (sumoylation of) the enzyme. Another study suggested moreover, that a N-terminal fragment of topo I is fully capable of undergoing nucleolar clearance in response to camptothecin despite being catalytically inactive (11Mao Y. Mehl I.R. Muller M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1235-1240Crossref PubMed Scopus (40) Google Scholar), which entirely contradicts our concept of catalytic enzyme-DNA interactions playing a role in this process. In summary, three mechanisms have been proposed for the partitioning of topo I between nucleoli an nucleoplasm: (i) (dis-)attachment to/from nucleolar sites of rDNA transcription, (ii) differences in enzyme mobility that are linked to DNA-catalysis, and (iii) a directed nucleolar import/export mechanism controlled by sumoylation of the enzyme. To determine which of these mechanisms is most appropriate, we have investigated here the nucleolar/nucleoplasmic partitioning of various biofluorescent topo I constructs that are active or not, and that contain the major sumoylation site or not. Moreover, we have compared the impact of the topo I poison camptothecin and the RNA polymerase inhibitor actinomycin D on the localization of these constructs. Constructs and Cell Culture—Construction and characterization of cell lines supporting stable expression of GFP-topo I, GFP-topo IPhe-723, GFP-topo I190–765, and GFP-topo I1–215 has been documented in previous studies (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Christensen M.O. Barthelmes H.U. Boege F. Mielke C. Nucleic Acids Res. 2003; 31: 7255-7263Crossref PubMed Scopus (13) Google Scholar). Please note that in one of these studies (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) GFP-topo I1–215 has been incorrectly referred to as GFP-topo I1–210, leading to the misunderstanding that it has five residues less than it actually does. Silencing of the major sumoylation sites of topo I (14Rallabhandi P. Hashimoto K. Mo Y.Y. Beck W.T. Moitra P.K. D'Arpa P. J. Biol. Chem. 2002; 277: 40020-40026Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) was done by site-directed mutagenesis using PCR primer pairs encoding the desired nucleotide exchanges, thereby generating GFP chimera of full-length human topo I with Lys to Arg point mutations at positions 117 (GFP-TIK117R), 103 and 117 (GFP-TIK103R,K117R), or 103, 117, and 153 (GFP-TIK103R,K117R,K153R). The active site mutant (GFP-TIPhe-723) was modified to express triple K103R,K117R,K153R point mutations by replacing a 3′-terminal restriction fragment of the topo I reading frame with the corresponding fragment of GFP-TIK103R,K117R,K153R, thereby generating GFP-TIPhe-723,K103R,K117R,K153R. To fuse GFP to the C terminus of the N-terminal domain of topo I as described previously (16Mao Y. Okada S. Chang L.S. Muller M.T. Cancer Res. 2000; 60: 4538-4543PubMed Google Scholar), the N-terminal topo I fragment (TI1–215) was inserted into the vector pMC-EGFPP (17Christensen M.O. Larsen M.K. Barthelmes H.U. Hock R. Andersen C.L. Kjeldsen E. Knudsen B.R. Westergaard O. Boege F. Mielke C. J. Cell Biol. 2002; 157: 31-44Crossref PubMed Scopus (181) Google Scholar) by linker-PCR, giving rise to the construct TI1–215-GFP. All new constructs were checked by DNA sequencing and stably expressed in the human embryonal kidney cell line HEK 293 (German Collection of Microorganisms and Cell Culture, Braunschweig, Germany) as described previously (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Christensen M.O. Barthelmes H.U. Boege F. Mielke C. Nucleic Acids Res. 2003; 31: 7255-7263Crossref PubMed Scopus (13) Google Scholar). Like in these previous studies, we have ascertained that all constructs are not overexpressed in relation to endogenous topo I, that the chimeric genes are not rearranged, and that green fluorescence of the cells could be unambiguously assigned to constitutive expression of the intended proteins. For inhibition of RNA polymerase I transcription, cells were incubated with 0.05 μg ml–1 actinomycin D. For induction of topo I cleavage complexes, cells were incubated with 20 μm camptothecin. Immunoblotting of Topoisomerase I—Cells in suspension were cultured in the absence or presence of 20 μm camptothecin for 20 min at 37 °C. For the detection of SUMO conjugates, cells were subjected to alkaline lysis and nuclease digestion as described (18Desai S.D. Li T.K. Rodriguez-Bauman A. Rubin E.H. Liu L.F. Cancer Res. 2001; 61: 5926-5932PubMed Google Scholar, 19Desai S.D. Liu L.F. Vazquez-Abad D. D'Arpa P. J. Biol. Chem. 1997; 272: 24159-24164Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). For band depletion assays, whole cell lysates were prepared by adding an equal volume of 2-fold lysis buffer (25 mm Tris-HCl, pH 6.8, 10% SDS, 8 m urea, 20% glycerol, 0.04% bromphenol blue, 10 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mm phenylmethylsulfonyl fluoride, 20 μg ml–1 aprotinin, 10 μg ml–1 pepstatin A). Material equivalent to 5 × 105 cells was then applied to each slot of an SDS-gel (9% (Fig. 1B) or 5.5% polyacrylamide (Fig. 2, A and B)). After electrophoresis, proteins were electroblotted onto polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford, MD). The membranes were subsequently blocked with PBS containing 2% BSA and 0.05% Tween 20, and then incubated for 1 h with mouse monoclonal antibodies against GFP (Clontech, Heidelberg, Germany) diluted with the same buffer. After washing, the filters were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG diluted with PBS containing 2% BSA and 0.1% Tween 20. Following extensive washing with the same buffer, labeled protein bands were visualized with the ECL Plus system (Amersham Biosciences, Freiburg, Germany).Fig. 2Topoisomerase I localization is not affected by mutational silencing of the major sumoylation sites.A, camptothecin-induced nucleolar de-localization of catalytic active topo I. Epifluorescent images of living cells expressing GFP chimera of full-length human topo I (GFP-TopoI) and point mutants of the major sumoylation sites K117R (GFP-TIK117R), K103R,K117R (GFP-TIK103R,K117R), and K103R,K117R, K153R (GFP-TIK103R,K117R,K153R) were acquired before (middle left) and after (middle right) exposure to camptothecin (20 μm CPT, 5 min). Corresponding phase contrast images are shown on the left. Camptothecin-induced sumoylation of GFP-topo I was assessed by GFP-directed immunoblotting (right). Cells untreated (lane 1) or treated with camptothecin (20 μm CPT, 20 min) (lanes 2 and 3) were subjected to alkaline lysis and then neutralized. In lane 3, samples were incubated in addition with Staphylococcus nuclease S7 to release topo I from covalent DNA-linkage. Unmodified GFP-topo I is marked by arrows and sumoylated enzyme moieties are marked by asterisks. B, sumoylation has no effect on the nuclear distribution of catalytic inactive topo I. Cells expressing GFP-chimera of wildtype topo I (GFP-TopoI), the active site mutant (GFP-TIPhe-723), or the active site mutant also bearing a triple-point mutation of the three major sumoylation sites (GFP-TIPhe-723,K103R,K117R,K153R) were cultured under the microscope, and phase contrast images (top) and images of corresponding green fluorescence (middle) were recorded. Sumoylation (bottom) was assessed as in A.View Large Image Figure ViewerDownload (PPT) Life Cell Imaging—For confocal imaging and fluorescent recovery after photobleaching (FRAP), we used a Zeiss LSM 510 inverted confocal laser scanning microscope equipped with a CO2-controlled on-stage heating chamber and a heated 63×/1.4 numerical aperture oil-immersion objective. Culturing of cells at 37 °C under the microscope was crucial for obtaining consistent data of localization and mobility of topo I, whereas erratic results were obtained, when native cell specimen were analyzed at ambient temperature (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). For FRAP measurements, fluorescent images of a single optical section were taken at 1.6-s time intervals before (n = 5) and after bleaching of a circular area at 20-milliwatt nominal laser power with three iterations. Imaging scans were acquired with the laser power attenuated to 0.1–1% of the bleaching intensity. For quantitative analysis of FRAP, fluorescence intensities of the bleached region and the entire cell nucleus were measured at each time point. Data were corrected for extracellular background intensity and for the overall loss in total intensity as a result of the bleach pulse itself and of the imaging scans. The relative intensity of the bleached area, Irel, was calculated according to a previous study (20Phair R.D. Misteli T. Nature. 2000; 404: 604-609Crossref PubMed Scopus (964) Google Scholar). For epifluorescence imaging of living cells at 37 °C, we used a Zeiss Axiovert 100 inverted microscope equipped with an on-stage heating chamber (ΔTC3 from Bioptechs, Butler, PA), a heated 63×/1.4 numerical aperture oil-immersion objective system, a cooled charge-coupled device camera (Sensys, Photometrics Ltd., München, Germany), and an additional 4-fold magnification lens. Immunohistochemistry—For immunostaining of RNA polymerase I, cells were grown on poly-l-lysine-coated microscopic glass slides, permeabilized with 0.07% Triton X-100 in PBS for 30 s at 37 °C, and then fixed with PBS containing 2% Paraformaldehyde (15 min, 4 °C). All subsequent steps were carried out at ambient temperature. After washing with PBS, the cell samples were blocked for 1 h with PBS supplemented with 2% BSA and 5% goat serum. To stain RNA polymerase I, the cell samples were then incubated for 1 h with human autoimmune serum S18 (21Reimer G. Rose K.M. Scheer U. Tan E.M. J. Clin. Invest. 1987; 79: 65-72Crossref PubMed Scopus (190) Google Scholar) diluted 1:800 in PBS. After washing with PBS, bound antibodies were visualized by incubation for 1 h with Cy3™-conjugated goat anti-human F(ab′)2 fragments (Dianova, Hamburg, Germany) diluted 1:1000 in PBS. Characteristics of the Experimental Setup—The topo I constructs outlined in Fig. 1A were constitutively expressed as N-terminal GFP fusions in human 293 cells (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 12Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). To determine the catalytic activity of the constructs in the cell, we assessed camptothecin-inducible immunoband depletion (22Liu L.F. Annu. Rev. Biochem. 1989; 58: 351-375Crossref PubMed Scopus (1920) Google Scholar). Before immunoblotting, cells were treated with camptothecin, which stabilizes covalent topo I·DNA intermediates. Because these intermediates are too large to enter the gel, the active fraction of the enzyme becomes depleted from the blot. As summarized in Fig. 1B, full-length topo I (GFP-topo I) became efficiently depleted (compare lanes 1 and 2), as did topo I lacking the first 190 amino acids (GFP-TI190–765, lanes 5 and 6). In contrast, the active site mutant (GFP-TIPhe-723, lanes 3 and 4) and the N-terminal domain alone (GFP-TI1–215, lanes 7 and 8) were not depleted. Using these constructs, we were thus able to compare the cellular behavior of the full-length human enzyme (GFP-topo I), retaining the major sumoylation site (Fig. 1A, black box) and being catalytically active in the cell, with two constructs also retaining the major sumoylation site but not being active in the cell (GFP-TIPhe-723 and GFP-TI1–215), and with a construct being active in the cell but lacking the major sumoylation site (GFP-TI190–765). Camptothecin-induced Nucleolar Depletion of Topoisomerase I Depends on Catalytic Activity but Not on the N-terminal Domain of the Enzyme—Fig. 1C shows from top to bottom representative fluorescence images of cells expressing GFP-topo I, GFP-TIPhe-723, GFP-TI190–765, or GFP-TI1–215 and their response to camptothecin treatment. The cells were cultured under the confocal microscope during observation. Each row shows the same cell, which was first imaged by transmitted light (left) and confocal sectioning of GFP-fluorescence at mid plane (middle). Then, cell culture was maintained for 5 min in the presence of 20 μm camptothecin, and the same cell was visualized again by confocal sectioning of green fluorescence (right). It can be seen that, in the absence of camptothecin, constructs GFP-topo I, GFP-TI190–765, and GFP-TI1–215 all accumulated in the nucleoli, whereas GFP-TIPhe-723 showed a more equal partitioning between nucleoli and nucleoplasm, which has previously been attributed to a lesser mobility of the mutant in the nucleoplasm (12Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). As that may be, Fig. 1C clearly shows that nucleolar/nucleoplasmic partitioning of topo I is independent of catalytic activity, because GFP-TI1–215 (inactive) and GFP-TI190–765 (active) both accumulated in the nucleoli in the same fashion as full-length topo I (GFP-topo I). As previously described (5Christensen M.O. Barthelmes H.U. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 35932-35938Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and also notable here in Fig. 1C, GFP-topo I and GFP-TI1–215 accumulate at the fibrillar centers of the nucleolus, whereas GFP-TI190–765 exhibits a more homogeneous nucleolar pattern. Fig. 1C demonstrates also that the construct lacking most of the N-terminal domain (GFP-TI190–765) was cleared from the nucleoli in response to camptothecin to the same extent as full-length topo I (GFP-topo I), whereas the catalytically inactive constructs GFP-TIPhe-723 and GFP-TI1–215 were both not cleared from the nucleoli. Thus, nucleolar clearance of topo I in response to camptothecin seems to occur irrespective of the presence of the first 190 amino acids, whereas it clearly requires catalytic activity. Silencing of the Major Sumoylation Sites Neither Affects Nucleolar Localization of topo I nor Its Nucleolar Depletion in Response to Camptothecin—The results shown in Fig. 1C indicate catalytic activity as the sole determinant of nucleolar depletion of topo I in response to camptothecin, which is in clear contrast to previous observations by others suggesting sumoylation of lysine residues 103, 117, and 153 to play a crucial role in this process (14Rallabhandi P. Hashimoto K. Mo Y.Y. Beck W.T. Moitra P.K. D'Arpa P. J. Biol. Chem. 2002; 277: 40020-40026Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This contradiction could be due to the fact that we have investigated here huge truncations of topo I, whereas the conflicting data were obtained by silencing just the relevant sumoylation sites in the full-length enzyme by point mutations. To exclude such truncation artifacts, we have repeated the experiments with cell lines stably expressing GFP fusions of full-length topo I, in which one (GFP-TIK117R), two (GFP-TIK103R,K117R), or all three major sumoylation sites (GFP-TIK103R,K117R,K153R) described previously (14Rallabhandi P. Hashimoto K. Mo Y.Y. Beck W.T. Moitra P.K. D'Arpa P. J. Biol. Chem. 2002; 277: 40020-40026Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) were silenced by point mutations. Fig. 2A shows images of these cells cultured at 37 °C under the microscope during observation. GFP fluorescence of the same set of cells is shown before (0′-CPT) and after culturing for 5 min in the presence of 20 μm camptothecin (5′-CPT). In the absence of camptothecin, all constructs accumulated in the nucleoli and concentrated at the nucleolar fibrillar centers, irrespective of the point mutations silencing the sumoylation sites (Fig. 2A, middle left column). It should also be noted that silencing the sumoylation sites did not disrupt binding of the enzyme to the nucleoli organizer regions of the akrocentric chromosomes during mitosis (data not shown). Thus, modification of topo I at the three major sumoylation sites seems not to affect topo I localization in the absence of camptothecin. Most notably, we found that GFP-TIK117R, GFP-TIK103R,K117R, and GFP-TIK103R,K117R,K153R were cleared from nucleoli upon exposure to camptothecin in the same fashion as the nonmutated enzyme GFP-topo I (Fig. 2A, middle right column). In keeping with the data obtained with the deletion mutant GFP-TI190–765 (Fig. 1C), these observations suggest that sumoylation at the three major acceptor sites does not play a role in nucleolar depletion of topoisomerase I in response to camptothecin. To demonstrate that GFP-topo I becomes indeed sumoylated in response to camptothecin in the cell line used in this study and that sumoylation is abolished by silencing the major acceptor sites, we subjected the cells to alkaline lysis followed by GFP-directed immunoblotting (Fig. 2A, right column). All constructs were catalytic active, as evidenced by depletion of the GFP-linked topo I (compare lanes 1 and 2). S7 nuclease treatment of the neutralized cell lysate released a ladder of evenly spaced, slower migrating SUMO conjugates of GFP-topo I (right column, top, lane 3, asterisks). The incidence of SUMO conjugates was drastically reduced by silencing of Lys-117 (compare lanes 3 in top and middle top panel), and it was completely abolished by silencing of two or all three major sumoylation sites (lanes 3 in middle bottom and bottom panel). Thus, we ascertained in the cell line employed in this study that GFP-topo I is sumoylated in response to camptothecin and that sumoylation is abolished by mutational silencing of the major sumoylation target sites described previously (14Rallabhandi P. Hashimoto K. Mo Y.Y. Beck W.T. Moitra P.K. D'Arpa P. J. Biol. Chem. 2002; 277: 40020-40026Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 23Horie K. Tomida A. Sugimoto Y. Yasugi T. Yoshikawa H. Taketani Y. Tsuruo T. Oncogene. 2002; 21: 7913-7922Crossref PubMed Scopus (52) Google Scholar). Recently, it has been shown that the active site mutant topo IPhe-723 is permanently sumoylated in a camptothecin-independent manner (23Horie K. Tomida A. Sugimoto Y. Yasugi T. Yoshikawa H. Taketani Y. Tsuruo T. Oncogene. 2002; 21: 7913-7922Crossref PubMed Scopus (52) Google Scholar). To test whether this accounts for its aberrant nuclear/nucleolar partitioning (Fig. 1C and Ref. 12Christensen M.O. Barthelmes H.U. Feineis S. Knudsen B.R. Andersen A.H. Boege F. Mielke C. J. Biol. Chem. 2002; 277: 15661-15665Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), we silenced the three major sumoylation sites in addition to the active site. The resulting quadruple mutant GFP-TIPhe-723,K103R,K117R,K153R was not sumoylated (Fig. 2B, bottom right), as opposed to the active site mutant GFP-TIPhe-723, which exhibited the expected, camptothecin-independent type of sumoylation (Fig. 2B, bottom middle). Thus, silencing of the major sumoylation sites does not restore proper nucleolar positioning to the active site mutant of topo I (compare right and middle images in the top section of Fig. 2B), and its aberrant nuclear/nucleolar partitioning cannot be attributed to constitutive sumoylation at these sites. In summary, the data in Fig. 2 rule out that sumoylation of amino acids Lys-103, Lys-117, and Lys-153 of topo I determines the partitioning of the enzyme between nucleoli and nucleoplasm or triggers its nucleolar clearance in response to camptothecin. On the contrary, these data suggest that sumoylation and nucleolar clearance of topo I occur independent of each other, although both effects are induced by camptothecin. Actinomycin D-induced Nucleolar Relocation of Topoisomerase I Requires the N-terminal Domain of the Enzyme but Not Its Catalytic Activity—To determine the role of ongoing rDNA transcription in the nuclear positioning of topo I, we monitored cells expressing GFP-topo I, GFP-TIPhe-723, GFP-TI190–765, or GFP-TI1–215 under a confocal laser scanning microscope, added actinomycin D to the culture medium at concentrations sufficient to completely arrest rDNA transcription (10Perry R.P. Exp. Cell Res. 1963; 29: 400-406Crossref Scopus (198) Google Scholar) and took serial confocal scans every 20 min (Fig. 3A). The first column shows full-length topo I (GFP-topo I). The topmost image (0′-ActD) was recorded immediately before adding actinomycin D. It shows the normal nucleolar localization of the enzyme, which remained largely unaltered during the first 20 min of exposure to the drug (compare 0′-ActD with 20′-ActD). How

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