Changes in Mobility Account for Camptothecin-induced Subnuclear Relocation of Topoisomerase I

Abstract

DNA topoisomerase I is a nucleolar protein, which relocates to the nucleoplasm in response to drugs stabilizing topoisomerase I⅐DNA intermediates (e.g. camptothecin). Here we demonstrate that this phenomenon is solely caused by the drug's impact on the interplay between mobility and localization of topoisomerase I in a living cell nucleus. We show by photobleaching of cells expressing biofluorescent topoisomerase I-chimera that the enzyme moves continuously between nucleoli and nucleoplasm. Complex kinetics of fluorescence recovery after photobleaching indicates that two enzyme fractions with different mobility coexist in nucleoli and nucleoplasm. However, the whole complement of topoisomerase I is in continuous flux between these compartments and nucleolar accumulation can plausibly explained by the enzyme's 2-fold lesser overall mobility in nucleoli versus nucleoplasm. Upon addition of camptothecin, topoisomerase I relocates within 30 s from the nucleoli to radial nucleoplasmic structures. At these sites, the enzyme becomes retarded in a dose-dependent manner. Inside nucleoli the mobility of topoisomerase I is much less affected by camptothecin. Thus, the enzyme's distribution equilibrium is shifted toward the nucleoplasm, which causes nucleolar delocalization. In general, topoisomerase I is an entirely mobile nuclear component, unlikely to require specific signaling for movements between nuclear compartments. DNA topoisomerase I changes the pitch of DNA helices by cutting one DNA strand and allowing passage of the complementary strand through the transient nick (1). One important role for this mechanism is the release of torsional stress created by DNA transcription. Thus, topoisomerase I activity is in principle required in the nucleoplasm for mRNA synthesis (2) and in the nucleolus for rRNA synthesis (3). However, the latter is considered the major working place of the enzyme, which has been recognized as a predominantly nucleolar protein (3, 4). Camptothecin and other agents stabilizing covalent topoisomerase I⅐DNA intermediates cause relocation of topoisomerase I from the nucleoli to the nucleoplasm (5). This was also observed with inhibitors of RNA synthesis prompting the conclusion that the phenomenon might be related to reduced activity of rRNA biosynthesis in the nucleolus (6). Alternatively, it has been proposed that camptothecin-induced conjugation of topoisomerase I with small ubiquitin-like modifiers might serve as a signal triggering translocation of topoisomerase I from the nucleoli to the nucleoplasm (7).Until now the dynamic properties of topoisomerase I in the nucleus or between nuclear compartments have not been assessed directly. Thus, it is not clear whether the enzyme is mobile or not, whether it resides in the nucleolus in a static manner and moves into the nucleoplasm in response to specific signals (as suggested in ref. 7) or whether it shuttles continuously between nucleolus and nucleoplasm. Consequently, it is also unclear as to what is the molecular event that leads to nucleolar del...

ATM, the Mre11/Rad50/Nbs1 complex, and topoisomerase I are concentrated in the nucleus of Purkinje neurons in the juvenile human brain

“…This pattern is consistent with other data showing that TOP1 is primarily localized to the nucleolus [23], but shuttles between the nucleolus and the nucleoplasm [24,25], and therefore provides further evidence of the specificity of staining. In some Purkinje neurons however, this pattern was reversed, with the amount of TOP1 being lower in the nucleolus than in the nucleoplasm ( Figure 5B, bottom row).…”

“…Also, our localization of the MRN proteins in human Purkinje neurons is completely consistent with the observations of Jacobsen et al [19]. With regard to TOP1, in addition to obtaining identical results using two independently generated monoclonal antibodies, our observation that TOP1 is localized to the nucleolus and nucleoplasm is consistent with other studies on the distribution of this enzyme within the nucleolus [23], and nucleus, including observations of GFP-tagged TOP1 in living human cells [24,25]. It should be noted that in one study [34], cytoplasmic TOP1 staining was observed in the mouse Purkinje neurons.…”

The N-terminal Domain Anchors Human Topoisomerase I at Fibrillar Centers of Nucleoli and Nucleolar Organizer Regions of Mitotic Chromosomes

“…However, we have recently observed that in a living cell topoisomerase I exchanges between nucleoli and the nucleoplasm in a rapid and continuous manner, suggesting that the distribution of the enzyme between these compartments is more likely governed by differences in residence time than by directing signals (2). In keeping with this, we show here that nucleolar accumulation of various truncated versions of topoisomerase I is correlated with their lesser mobility in this compartment and not with the N-terminal portion being intact or present.…”

“…In keeping with this, studies of genomic DNA cleavage by topoisomerase I have invariantly come up with sites in the rDNA (3)(4)(5). However, catalytic interactions with rDNA cannot per se account for the accumulation of topoisomerase I in the nucleoli because stabilization of covalent topoisomerase I⅐DNA intermediates by camptothecin immobilizes the enzyme preferentially at nucleoplasmatic sites and only to a much lesser extent in the nucleoli (2). Along this reasoning the question arises of what restricts topoisomerase I in the nucleolus and how is this phenomenon related to rDNA processing.…”

“…However, the latter task seems to dominate. During interphase, most of topoisomerase I is located in the nucleoli and not in the nucleoplasm, although the enzyme interchanges constantly between these two compartments (2). It has been proposed that nucleolar accumulation of topoisomerase I reflects engagement in rDNA-transcription because the rRNA genes are by far the most heavily transcribed genes in the cell.…”