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Originally published In Press as doi:10.1074/jbc.M400498200 on March 9, 2004

J. Biol. Chem., Vol. 279, Issue 21, 21873-21882, May 21, 2004
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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*

Morten O. Christensen{ddagger}, René M. Krokowski{ddagger}, Hans U. Barthelmes{ddagger}, Robert Hock§, Fritz Boege{ddagger}, and Christian Mielke{ddagger}

From the {ddagger}Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich Heine University, Medical School, Moorenstrasse 5, D-40225 Duesseldorf and the §Department of Cell and Developmental Biology, Biocenter, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany

Received for publication, January 16, 2004 , and in revised form, February 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (<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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanism that distributes DNA-topoisomerase I (topo I)1 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 (1). 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 (2) and rDNA (3, 4). 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 (5). 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 (6). 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 (5). 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 (7, 8). Because such compounds also inhibit RNA synthesis (9) and because direct inhibitors of RNA polymerase I such as actinomycin D (10) 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 (8, 11). 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 (12). However, this simple explanation was disputed, because camptothecin also stimulates modification of topo I with small ubiquitin-like modifiers (SUMO) (13), 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 (14). 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 (11), 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (5, 12, 15). Please note that in one of these studies (5) 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 (14) 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 (16), the N-terminal topo I fragment (TI1–215) was inserted into the vector pMC-EGFPP (17) 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 (5, 12, 15). 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 (18, 19). 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 x 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).



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  		FIG. 1.
The impact of camptothecin on in vivo localization of various GFP-linked topo I constructs. A, topo I constructs. The putative sumoylation site, including flanking {psi}KXE sequences (residues 103, 117, and 153 (14)) is marked by a black box, whereas the GFP moiety appended at the N terminus is gray-shaded. B, topo I immuno-band depletion. Cell lines expressing GFP chimera of full-length human topo I (GFP-Topo I, lanes 1 and 2), the active site mutant (GFP-TIPhe-723, lanes 3 and 4), the N-terminally truncated enzyme (GFP-TI190–765, lanes 5 and 6), and the C-terminally truncated enzyme (GFP-TI1–215, lanes 7 and 8) were cultured either in plain medium (lanes 1, 3, 5, and 7) or in medium containing 20 µM camptothecin for 20 min (lanes 2, 4, 6, and 8) and then subjected to Western blotting using antibodies against GFP. Molecular weight values on the right margin were deduced from migration distances of marker proteins on the same gel. C, camptothecin-induced nucleolar de-localization. Confocal fluorescent images of living cells expressing GFP chimera of full-length human topo I (GFP-TopoI), the active site mutant (GFP-TIPhe-723), the N-terminally truncated enzyme (GFP-TI190–765), and the C-terminally truncated enzyme (GFP-TI1–215) are shown before (middle) and after (right) exposure to camptothecin (20 µM CPT, 5 min). Corresponding images obtained by transmitted light are shown on the left.

 



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  		FIG. 2.
Topoisomerase 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.

 
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 63x/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 (5). 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 (20). For epifluorescence imaging of living cells at 37 °C, we used a Zeiss Axiovert 100 inverted microscope equipped with an on-stage heating chamber ({Delta}TC3 from Bioptechs, Butler, PA), a heated 63x/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 (21) diluted 1:800 in PBS. After washing with PBS, bound antibodies were visualized by incubation for 1 h with Cy3TM-conjugated goat anti-human F(ab')2 fragments (Dianova, Hamburg, Germany) diluted 1:1000 in PBS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (5, 12). To determine the catalytic activity of the constructs in the cell, we assessed camptothecin-inducible immunoband depletion (22). 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 (12). 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 (5) 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 (14). 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 (14) 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 (14, 23). Recently, it has been shown that the active site mutant topo IPhe-723 is permanently sumoylated in a camptothecin-independent manner (23). To test whether this accounts for its aberrant nuclear/nucleolar partitioning (Fig. 1C and Ref. 12), 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 (10) 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). However, after culturing the cells for 40 min in the presence of actinomycin D (40'-ActD) significant changes in the nucleolar localization of GFP-topo I became apparent. Now, the enzyme was placed in large structures at the rim of the nucleoli (the cell shown in the first column of Fig. 3A at 40'-ActD exhibits three such structures). This pattern became even more distinct after 60 or 80 min of exposure to actinomycin D (Fig. 3A, column 1, 60'-ActD and 80'-ActD), when round or crescent-like structures at the rim of the nucleoli became a preeminent feature. Interestingly, the catalytically inactive and camptothecin-insensitive mutants GFP-TIPhe-723 and GFP-TI1–215 (Fig. 3A, second and fourth columns, respectively) responded to inhibition of RNA polymerase I in a similar fashion and with a similar time course as full-length topo I. Both constructs formed crescent or globular structures at the rim of the nucleoli after more than 40 min of exposure to actinomycin D (compare columns 1, 2, and 4 of Fig. 3A at 40'-ActD, 60'-ActD, and 80'-ActD). In contrast, the localization of the topo I construct lacking the first 190 amino acids (GFP-TI190–765) was hardly effected by actinomycin D (Fig. 3A, third column). Upon cross reference of these findings with the data shown in Fig. 1B, it becomes apparent that all constructs of topo I that retained the N-terminal domain were relocated to the nucleolar perimeter in response to actinomycin D irrespective of whether they were active (GFP-topo I) or not (GFP-TIPhe-723 and GFP-TI1–215), whereas the construct lacking a considerable portion of the N-terminal domain (GFP-TI190–765) was not affected by actinomycin D, although it was clearly active in the cell (i.e. given to camptothecin-induced immunoband depletion, as shown in Fig. 1B). Summing up, we concluded that actinomycin D and camptothecin are affecting the nucleolar positioning of topo I by independent mechanisms: actinomycin D induces relocation of topo I to the nucleolar perimeter by a mechanism that requires the presence of the N-terminal domain, but not catalytic activity. In contrast, camptothecin induces spreading of topo I to the entire nucleoplasm by a mechanism that requires catalytic activity but not the N-terminal domain of the enzyme.



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  		FIG. 3.
The impact of actinomycin D on in vivo localization of GFP-linked topo I, and its colocalization with RNA polymerase I. A, actinomycin D-induced localization changes. Cells expressing GFP chimera of full-length human topo I (GFP-TopoI), the active site mutant (GFP-TIPhe-723), the N-terminally truncated enzyme (GFP-TI190–765), and the C-terminally truncated enzyme (GFP-TI1–215) were cultured under a confocal microscope and exposed to 0.05 µgml–1 actinomycin D, and images were obtained after various time periods. Representative images of a cell are shown before (0'-ActD) and at the indicated time points after the addition of actinomycin D (20'-80'-ActD). For each cell line a corresponding transmitted light image (Tr. Light) before the addition of actinomycin D is shown on the top. Arrowheads mark the position of globular structures formed at the nucleolar rim after 80 min of exposure to actinomycin D. B, co-localization with RNA polymerase I. The same cell lines as in A were fixed and counterstained with antibodies against RNA polymerase I. Corresponding images of the same cell obtained by phase contrast (left), GFP fluorescence (middle), and antibody-derived fluorescence (right) are shown.

 
Actinomycin D-induced Nucleolar Relocation of topo I Correlates with the Co-localization of the Enzyme with RNA Polymerase I at Nucleolar Fibrillar Centers—We have recently shown that the N-terminal domain (the first 215 amino acids) of topo I serves as an adaptor that anchors a subpopulation of the enzyme at fibrillar centers of nucleoli, the location of rDNA and RNA polymerase I (5). Fig. 3A demonstrates that the same domain is required for the nucleolar clearance of topo I in response to actinomycin D, suggesting that the anchoring at fibrillar centers and the co-localization with RNA polymerase I might again be involved. To assess co-localization with RNA polymerase I, we counterstained cells expressing GFP-topo I, GFP-TIPhe-723, GFP-TI190–765, and GFP-TI1–215 with antibodies against RNA polymerase I (Fig. 3B). Upon comparing the GFP signal of full-length topo I (GFP-topo I, middle) with the immunofluorescent signal specific for RNA polymerase I (right), it becomes apparent that both proteins co-localize at the fibrillar centers. A similar observation was also made for the active site tyrosine mutant (GFP-TIPhe-723), which in the living cell has a more even distribution between nucleoli and nucleoplasm than the wild type enzyme and exhibits only a faint granular pattern inside the nucleoli (see Fig. 1C). However, the granular pattern inside nucleoli became enhanced by the immunostaining procedure, and it can be seen that, in these structures, GFP-TIPhe-723 co-localized with RNA polymerase I (Fig. 3B). A stringent co-localization with RNA polymerase I was also seen with the N-terminal domain alone (GFP-TI1–215), where GFP- and antibody-derived signals were almost identical. In contrast, topo I lacking the first 190 amino acids (GFP-TI190–765) did not concentrate at the fibrillar centers and did not co-localize there with RNA polymerase I. The N-terminally truncated construct was homogenously distributed through the entire nucleolar space (although we could not directly demonstrate its exclusion from fibrillar centers). In summary, these data imply that co-localization of topo I with the fibrillar centers and the RNA polymerase I holoenzyme via the N-terminal domain is a prerequisite for the nucleolar positioning of the enzyme at fibrillar centers and the relocation to the nucleolar perimeter upon inhibition of rDNA transcription. In keeping with this, incremental deletions revealed that the entire N-terminal domain of topo I is required for its restricted co-localization with RNA polymerase I, whereas even minor deletions at the N-terminal end result in a diffuse nucleolar distribution (5).

Camptothecin Attenuates Topoisomerase I Mobility in the Nucleoplasm, Whereas Actinomycin D Does Not—We have previously shown that camptothecin rapidly induces a retardation of topo I in the nucleoplasm (12). To exclude a similar effect for actinomycin D, we measured the mobility of GFP-linked topo I after prolonged exposure to actinomycin D. As shown in Fig. 4A, FRAP kinetics obtained in the nucleoplasm of untreated cells ({circ}) and cells treated for 60 min with actinomycin D ({triangleup}) were almost the same, whereas FRAP kinetics obtained in the nucleoplasm after exposure to camptothecin for 20 min ({square}) were markedly retarded. Thus, relocation of nucleolar topo I in response to actinomycin D does not involve attenuation of the enzyme in the nucleoplasm, whereas relocation in response to camptothecin does.



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  		FIG. 4.
The impact of actinomycin D on mobility and in vivo localization of GFP-linked topo I. A, FRAP-kinetics of GFP-linked topo I (GFP-TopoI) in the nucleoplasm of untreated cells ({circ}), or cells exposed to 0.05 µg ml–1 actinomycin D for 60 min ({triangleup}), or cells exposed to 20 µM camptothecin for 20 min ({square}). B, actinomycin D-induced changes in localization are overruled by camptothecin. Living cells expressing GFP-chimera of full-length human topo I (GFP-TopoI) were exposed to 0.05 µgml–1 actinomycin D for 60 min followed by exposure to 20 µM camptothecin for 5 min. Images of the same cell obtained by phase contrast (Phase) and GFP fluorescence before actinomycin D treatment (0'-ActD), after 60-min treatment (60'-ActD), and 5 min after addition of camptothecin (5'-CPT) are shown.

 
To study a superimposition of the two seemingly independent mechanisms, we first inhibited rDNA transcription with actinomycin D and then exposed the same cell to camptothecin. Fig. 4B shows a typical outcome of such an experiment performed on a living cell. Before addition of either drug (0'-ActD) the normal nucleolar pattern of topo I is exhibited, whereas after 60-min exposure to actinomycin D (60'-ActD) the same cell shows the dotted crescents along the nucleolar rims typical of prolonged inhibition of rDNA transcription (compare Figs. 4B and 3A). Upon subsequent addition of camptothecin (5'-CPT), topo I was rapidly redistributed from these dotted crescent structures to radial nucleoplasmic structures also induced by CPT in cells not pretreated with actinomycin D (compare Figs. 4B and 1C). This result demonstrates that cleavage activity of topo I is not affected by pre-treatment with actinomycin D, in as much as camptothecin still stabilizes the catalytic DNA intermediates of the enzyme, thereby retarding its mobility in the nucleoplasm. Consequently, the camptothecin effect overrides the actinomycin D effect. This further supports the notion, that the two drugs influence the localization of topo I by different mechanisms. Whereas the response to actinomycin D depends on topo I co-localization with the RNA polymerase I complex, the response to camptothecin solely requires catalytic activity of the enzyme and the stabilization of catalytic intermediates linked to genomic DNA.

During actinomycin D-induced Arrest of rRNA Synthesis, Topoisomerase I Remains Co-localized with RNA Polymerase I, and the Two Proteins Co-migrate to the Nucleolar Perimeter—In cells undergoing nucleolar inactivation after forced transcriptional arrest, segregation of nucleolar structures has been observed, which is characterized by a spatial dissociation of the fibrillar components (i.e. fibrillar centers and dense fibrillar component) from the granular component (24). Interestingly, in such segregated nucleoli RNA polymerase I remains attached to the fibrillar centers, thus giving rise to crescent-like structures at the nucleolar perimeter (21, 25), which are highly reminiscent of the ones seen here with topo I after prolonged exposure to actinomycin D (Fig. 3A). This analogy led us to speculate that topo I might actually remain co-localized with the RNA polymerase I holo-enzyme (and the fibrillar centers) during blockade of rDNA transcription by actinomycin D. Thus, it should be subjected to nucleolar relocation together with RNA polymerase I. To test this hypothesis, we compared morphology and time course of actinomycin D-induced nucleolar relocation of GFP-linked topo I with that of RNA polymerase I visualized in the same cells by immunostaining. Fig. 5A summarizes representative results obtained after various times of exposure to actinomycin D. The GFP signal of full-length topo I is shown in the middle, whereas corresponding immunofluorescent patterns specific for RNA polymerase I are shown on the right. Upon comparing the two columns of images, it becomes clear that before treatment (0'-ActD) both proteins co-localize at granular substructures of nucleoli representing the fibrillar centers (compare Fig. 3B). Very similar patterns were obtained in cells exposed to actinomycin D for 20 min (20'-ActD), whereas after 40 min of exposure to actinomycin D (40'-ActD) topo I started to relocate to crescent-like structures at the nucleolar perimeter, and these structures became even more pronounced after 60 min of exposure (60'-ActD), which is consistent with the corresponding time course shown in Fig. 3A (GFP-topo I). After 40 and 60 min of exposure to actinomycin D, RNA polymerase I exhibited crescent-like, peri-nucleolar structures that were exactly matching the GFP-signal of GFP-topo I, suggesting that it was redistributed exactly in the same fashion as topo I (Fig. 5A, compare middle and right). Thus, the two proteins relocate together to the nucleolar perimeter upon exposure to actinomycin D, and it can be concluded that topo I co-localizes with RNA polymerase I before, during, and after transcriptional arrest.



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  		FIG. 5.
The impact of camptothecin and actinomycin D on the localization of RNA polymerase I. Cells expressing GFP-TopoI were grown on microscope slides and treated either with 0.05 µg/ml actinomycin D (A) or 20 µM camptothecin (B). Before (0') and 20, 40, or 60 min after treatment, cells were fixed and stained with antibodies against RNA polymerase I. Representative images obtained by phase contrast (left), GFP-fluorescence (middle), and immunofluorescence (right) are shown.

 
Upon Camptothecin Exposure, Topoisomerase I and RNA Polymerase I Dissociate and Are Both Relocated Independently of Each Other—Because camptothecin has a similar potential of inhibiting rDNA transcription as actinomycin D (9), we wondered whether it would induce nucleolar relocation of RNA polymerase I in the same fashion as actinomycin D, and, if so, whether topo I and RNA polymerase I would again remain co-localized during such a process. To address this question, we repeated the experiment outlined in Fig. 5A with cells exposed to camptothecin instead of actinomycin D. These data are summarized in Fig. 5B. Before treatment (0'-CPT), topo I and RNA polymerase I co-localized in granular nucleolar structures in the usual manner (compare to Figs. 3B and 5A). However, after 20 min of exposure to camptothecin (20'-CPT), topo I was completely cleared from the nucleoli, whereas RNA polymerase I was still located at the fibrillar centers within the nucleoli. Longer exposure to camptothecin (40'-CPT and 60'-CPT) resulted in a relocation of RNA polymerase I to crescent-like structures at the nucleolar perimeter similar to those observed after actinomycin D treatment. However, topo I and RNA polymerase I did not co-localize in such structures, as was the case after actinomycin D-treatment (Fig. 5, compare A and B). Thus, camptothecin exerts a dual effect on nucleolar structure and function. On the one hand, it has a chronic effect, which takes more than 20 min and is similar to that of actinomycin D, inasmuch as it comprises nucleolar segregation and redistribution of RNA polymerase I into crescent-like structures at the nucleolar perimeter. On the other hand, it has an immediate effect, which is characterized by an immediate dissociation of topo I from the RNA polymerase I holo-enzyme at the nucleolar fibrillar centers and retardation of the enzyme in the nucleoplasm.

Long-termed Treatment with Camptothecin Induces Nucleolar Depletion of the N-terminal Domain of Topoisomerase I Due to Nucleolar Segregation—It has been reported that the catalytic inactive N-terminal domain of topo I alone is cleared from the nucleoli in response to camptothecin (11), whereas we find here that the phenomenon relies on catalytic activity of the enzyme in a crucial manner and, thus, is not observed with the N-terminal domain alone (Fig. 1C). One possible explanation of this discrepancy could be the different type of GFP fusion used here and in the previous study. Another, more plausible reconciliation could be gained from the data in Fig. 5B, showing that early on (after less than 5 min) camptothecin retards topo I in the nucleoplasm, which results in a rapid redistribution of active enzyme to the nucleoplasm. However, later on (after more than 20 min), camptothecin apparently also induces nucleolar segregation, which should lead to a delayed nucleolar clearance of all those forms of the enzyme that are inactive but retain the N-terminal domain, criteria that would apply to any construct of the N-terminal domain alone. To test this hypothesis, we subjected cells expressing the N-terminal domain of topo I fused to GFP either at the N terminus (Fig. 6, top, GFP-TI1–215) or at the C terminus (Fig. 6, bottom,TI1–215-GFP) to prolonged treatments with camptothecin. Upon comparing images of GFP-fluorescence obtained before (0'-CPT) and 5 min after exposure to camptothecin (5'-CPT), it becomes quite clear that both constructs were located in granular structures of the nucleolus and did not exhibit a rapid relocation to the nucleoplasm in response to camptothecin, which supports the results shown in Fig. 1C and excludes artifacts due to different orientations of the GFP fusion. Upon prolonged exposure to camptothecin (40'-CPT), however, both constructs were relocated to crescent-like structures, equivalent to those found after nucleolar segregation in response to actinomycin D (compare Figs. 6 to 5A). Taking into account that in the previous study (11) exposure times to camptothecin were usually 30 min, it can well be imagined that these authors studied slow relocation of topo I due to inhibition of RNA synthesis and nucleolar segregation, which is shared by all active and inactive forms of the enzyme that retain the entire N-terminal domain, whereas we studied rapid relocation due to prolonged binding of topo I to genomic DNA, which is shared by all active forms of the enzyme irrespective of the presence of the N-terminal domain.



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  		FIG. 6.
Camptothecin-induced transcriptional arrest changes the localization of catalytic inactive GFP-linked constructs of the N-terminal domain of topo I. Green fluorescence of living cells expressing the C-terminally truncated enzyme fused to GFP at its N terminus (GFP-TI1–215) or its C terminus (TI1–215-GFP) was imaged before (0'-CPT), after 5 min (5'-CPT), or after 40 min (40'-CPT) exposure to 20 µM camptothecin. For each cell a corresponding phase-contrast image before the addition of camptothecin is shown on the left.

 
Camptothecin-induced Nucleolar Depletion of Topoisomerase I Is Rapidly Reversible and Independent of Nucleolar Segregation, Which Is Slowly Reversible—Camptothecin stabilizes catalytic covalent DNA intermediates of topo I in a reversible manner, and it has been demonstrated in vitro and in vivo that such complexes will readily disappear upon removal of the drug (26–28). Because the data presented here and in our previous investigation (12) strongly suggest that nucleolar clearance of topo I in response to camptothecin is closely related to the stabilization of the enzyme in covalent DNA intermediates in the nucleoplasm, we wondered whether reversal of such complexes upon removal of the drug would also lead to a reversal of the nucleoplasmic spreading of the enzyme and to its reappearance at the usual nucleolar position. To test this, we cultured cells expressing GFP-linked topo I under a confocal laser scanning microscope and observed changes in its cellular localization in response to a brief incubation (2 min) with camptothecin, followed by removal of the drug. The first image (Fig. 7A, 0'-CPT) was recorded immediately before adding camptothecin and shows the enzyme at its normal nucleolar position, which is rapidly lost as the enzyme is redistributed to the nucleoplasm upon exposure to camptothecin (Fig. 7A, 2'-CPT). The next image obtained 5 min after removal of the drug (Fig. 7A, 5'-Wash) demonstrates the onset of a rapid repositioning of topo I to the fibrillar centers inside the nucleolus, which is complete after 10 min (Fig. 7A, 10'-Wash). However, when the cells were treated with camptothecin for longer periods (60 min) before removing the drug (Fig. 7B), then topo I did not re-enter the nucleolus (compare 5'-Wash in Fig. 7, A and B) but rather accumulated in crescent-like structures at the nucleolar rim, which are reminiscent of the localization of the enzyme seen after prolonged exposure to actinomycin D (see Fig. 3A, GFP-topo I). These structures were maintained for at least 30 min following removal of camptothecin (Fig. 7B, 10–30'-Wash), and the usual granular nucleolar pattern inside the nucleoli was not completely re-established until 60 min after removal of the drug (Fig. 7B, 60'-Wash). These data show that the immediate effect of camptothecin on the nucleolar localization of topo I (i.e. dissociation from the fibrillar nucleolar centers and spreading to the nucleoplasm) is readily reversible. After removal of the drug, the enzyme will always rapidly and faithfully re-associate with the fibrillar centers, no matter if these are located inside the nucleoli (Fig. 7A) or at the nucleolar rim due to a segregation of the nucleolar structures after silencing of rDNA transcription (Fig. 7B). Thus, relocation of topo I in response to camptothecin is a mechanism entirely independent of the silencing of rDNA transcription and the nucleolar segregation of the rRNA transcription machinery, which are also effected by the drug upon prolonged exposure. Camptothecin-induced relocation of topo I is rapid. It has an onset time of less than 2 min and reverts within 10 min (Fig. 7A). Camptothecin-induced nucleolar segregation is slow. It has an onset time of 20–40 min (Fig. 5A) and a reversal time of 40–60 min (Fig. 7B). It should be noted that upon prolonged exposure to camptothecin the slow mechanism appears to act on top of the fast one.



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  		FIG. 7.
Reversibility of the camptothecin-effect on topo I localization. Cells expressing GFP chimera of full-length human topo I (GFP-TopoI) were cultured under a confocal microscope and treated with camptothecin for 2 min (A) or 60 min (B). Cells were then washed several times with warm medium to remove camptothecin and culture was continued in the absence of the drug for up to 60 min. Time-lapsed images of the same individual cell were recorded before (0'-CPT) and after addition of 20 µM camptothecin (2'-CPT and 60'-CPT) and after various time periods after washing out the drug (5'- and 60'-Wash). To identify changes in nucleolar structures, a corresponding transmitted light image is shown for each observation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Partitioning of Topoisomerase I between Nucleoli and Nucleoplasm—This partitioning is an issue, because it provides a morphological criterion for the distribution of the enzyme between nucleolar and nucleoplasmic tasks, and thus may help to elucidate the general mechanism by which the cellular complement of topo I is appropriately assigned to processes as diverse as mRNA transcription (2), rRNA transcription (3, 4), DNA synthesis (29), DNA repair (30), and DNA recombination (31), all of which require topo I activity as a cofactor. Several investigators have studied the distribution mechanism by comparing the effects of topo I inhibitors and other inhibitors of rRNA synthesis on the sub-nuclear localization of topo I. However, the results were equivocal and have suggested at least three different mechanisms that could target topo I to nucleolar functions: (i) recruitment to sites of active rRNA transcription (7, 8), (ii) accumulation at sites of lesser mobility (12), and (iii) active transport into and out of the nucleolus that is governed by attachment of small ubiquitin-like modifiers to a certain site within the N-terminal domain of topo I (14, 32). This controversy led us to re-investigate the matter using constitutive expression of various bio-fluorescent constructs of topo I as a means for monitoring the nucleolar/nucleoplasmic partitioning of the enzyme in a living cell, and for assessing the role that is played in this by ongoing rRNA synthesis, enzyme activity, the major acceptor sites for sumoylation, and the N-terminal domain of the enzyme. We present evidence showing that nucleolar/nucleoplasmic partitioning of topo I is governed by a dynamic cooperation of the first two mechanisms mentioned above, whereas we could not find evidence for the third one to play a role in this.

The positioning of topo I within the nucleus is mostly determined by its association with the fibrillar centers, nucleolar structures containing the rDNA and a cluster of proteins required for rDNA transcription termed the RNA polymerase I holo-enzyme (33). Fibrillar centers are recently shown to be the nucleolar compartment where rDNA transcription takes place (6). Topo I is most likely a component of this cluster, because it co-purifies with RNA polymerase I (34) and co-localizes with it at granular substructures of the nucleolus (5). Until now, it was believed that this feature of topo I is linked to active rDNA transcription, because blockade of RNA synthesis leads to nucleolar clearance of the enzyme (8). However, we show here quite clearly (Fig. 5A) that co-localization of topo I and RNA polymerase I is maintained during exogenous inhibition of RNA synthesis and that nucleolar clearance of topo I in response to RNA synthesis inhibitors just reflects the partaking of the enzyme in the segregation of the inactivated nucleolar structure (24). This behavior of topo I closely resembles that of RNA polymerase I itself (21, 25). Thus, topo I does not make an exception to the general rule that molecules are targeted to the nucleolus due to their interaction with one of the three nucleolar building blocks that are structured around rDNA and/or its transcripts (35–37). Even during mitosis, where rDNA is not transcribed, topo I remains associated with the corresponding chromosomal structures that harbor rDNA and RNA polymerase I (5, 38, 39). Thus, positioning of topo I is crucially determined by its co-localization with rDNA and RNA polymerase I, and this co-localization is maintained throughout the entire cell cycle irrespectively of where in the nucleus these complexes are localized and independently of ongoing rRNA transcription or active DNA turnover by topo I itself. This association is, however, subjected to dynamic exchanges with other locations of topo I in the nucleus. Therefore, it is readily disrupted when topo I becomes fixed by camptothecin to these other locations (Figs. 4B and 5B), and it is readily reformed upon release of such external linkages (Fig. 7). Most notably, topo I reconnects with the fibrillar centers irrespectively of whether these are active and located inside the nucleoli (Fig. 7A), or inactive and located at the nucleolar perimeter (Fig. 7B). In summary, these data suggest a combined mechanism for the spatial partition-ing of topo I: On the one hand, topo I is targeted by constitutive features such as its inclination to co-localize with fibrillar centers and RNA polymerase I, which is an intrinsic property of its N-terminal domain and does not rely on the catalytic properties of the enzyme. On the other hand, topo I is targeted by the interplay with DNA and factors such as camptothecin, which influence its catalytic cycle. Strikingly, all constructs of topo I that were catalytically active (GFP-topo I and GFP-TI190–765) were also capable of undergoing rapid nucleolar clearance (less than 5 min) in response to camptothecin, whereas all inactive constructs (GFP-TIPhe-723 and GFP-TI1–215), first changed their localization after camptothecin had induced nucleolar segregation after more than 20 min. This coincidence strongly argues in favor of our previous hypothesis (12) that rapid nucleolar clearance in response to camptothecin just reflects a selective immobilization of topo I in the nucleoplasm, which arises from the impact of camptothecin on the interplay of active topo I and DNA.

Nucleolar Targeting of Topoisomerase I Does Not Depend on the Presence of the Major Sumoylation Site—It has been shown that camptothecin stimulates modification of topo I with ubiquitin (18) and SUMO (13) and that these modifications play a crucial role in resistance of tumor cells to camptothecin, because they trigger proteolytic degradation of DNA-linked topo I (40). Thus, it is clear that both effects are important cellular responses to topo I-mediated DNA damage. However, the exact cellular consequences of the attachment of SUMO to topo I needs still to be clarified (41). Recently, it has been speculated that the same mechanism might regulate nucleolar import and export of topo I, because mutational silencing of the major target site for the modification with small ubiquitin-like modifiers enhances nucleolar accumulation of the enzyme and abolishes its nucleolar clearance in response to camptothecin (14). However, this hypothesis is not supported by the data presented here. We demonstrate that the truncated enzyme GFP-TI190–765 is rapidly cleared from nucleoli upon exposure to camptothecin to the same extent as full-length topo I, although it does not contain the sumoylation sites at residues 103, 117, and 153. Conversely, the active site mutant (GFP-TIPhe-723) and the N-terminal domain alone (GFP-TI1–215) are both not cleared from the nucleoli in response to camptothecin, although both contain the sumoylation sites. In keeping with this, we find that silencing of the major sumoylation sites by point mutations does not prevent camptothecin-induced nucleolar clearance of the enzyme (Fig. 2A). Thus, rapid nucleolar clearance of topo I occurs irrespective of the sumoylation site being present or not, and sumoylation at this site is unlikely to play a role in the clearing process of topo I. Nonetheless, sumoylation could play a general role in the distribution of topo I between nucleoli and nucleoplasm, even though it is not involved in the clearing process induced by camptothecin. We have observed previously that the ability of nucleolar accumulation is markedly impaired in the active site tyrosine mutant of topo I (12), which at the same time is sumoylated in a constant and camptothecin-independent manner (Fig. 2B and Ref. 23). This observation led us to speculate that sumoylation might impede access of topo I to the nucleoli. However, the data shown in Fig. 2B argue to some extent against this hypothesis, because they demonstrate that the ability of nucleolar accumulation cannot be restored to the active site mutant by abolishing its sumoylation. In summary, we conclude that sumoylation does not play a role in the distribution of topo I between nucleoli and nucleoplasm. The striking contrast between our findings and the results of Rallabhandi and coworkers (14) is difficult to explain. It could stem from differences between cell lines, because Rallabhandi et al. have used HeLa cells, whereas we have used 293 cells. However, 293 cells support sumoylation of topo I in response to camptothecin just as well as HeLa cells (Fig. 2 and Ref. 23). Another source of discrepancy could be that our data were obtained by observation of living cells cultured under the microscope, whereas Rhallabhandi and coworkers investigated fixed specimen. From our experience, such experimental details can have a rather pronounced and unpredictable effect on the nuclear distribution of topo I (5).

Our view of sumoylation and nucleolar clearance of topo I being two unrelated events both induced by camptothecin is also supported by biochemical data indicating that ubiquitinylation and sumoylation are targeted at topo I molecules bound to DNA. The formation of these cleavable complexes induced by CPT can be enhanced by overexpression of SUMO-1 and conversely reduced by expression of UBC9cs, a dominant negative mutant of the E2 SUMO transferase that acts as a suppressor of sumoylation (23). Thus, sumoylation seems a positive regulator of camptothecin-induced formation of topo I·DNA complexes (23), probably by increasing the half-life of such complexes. In contrast, free topo I does not appear to be a target for sumoylation (42), because nuclease treatment is necessary to visualize ubiquitin or SUMO conjugates of topo I by Western blotting (13, 19). Thus, a plausible sequence of events seems to be that camptothecin first stabilizes topo I on the DNA, which for quantitative reasons occurs more in the nucleoplasm than in the nucleolus (12), although topo I cleavage of rDNA is also stimulated by the drug (43–45). Subsequently, the DNA-linked enzyme becomes sumoylated (13). In such a scenario, sumoylation is less likely to effect topo I in the nucleolus, because there the enzyme is either less bound to DNA or less targeted by camptothecin, in as much as it is scarcely immobilized in this compartment by the drug (12).


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft (Grants Bo 910/3-1, Bo 910/3-2, Bo 910/4-1, Bo 910/5-1, GRK 639, and GRK 1033) is acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 49-211-811-9323; Fax: 49-211-811-8021; E-mail: christian.mielke{at}med.uni-duesseldorf.de.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We are grateful to Jörg Hacker and Hilde Merkert from the Research Center for Infectious Diseases, University of Würzburg, Germany, for generously providing access to their confocal laser-scanning microscope.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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