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

Abstract

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.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 typ...

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.…”

TDP1 Overexpression in Human Cells Counteracts DNA Damage Mediated by Topoisomerases I and II

“…These findings suggest that nucleolar clearance of TDP1 upon prolonged exposure to camptothecin does not stem from a comigration with topo I to the nucleoplasm but rather from the general disintegration and redistribution of nucleolar structures upon prolonged inhibition of rDNA transcription by camptothecin (25). This notion is also supported by the observation that exposure to the topo II-poison VP-16 did not induce nucleolar depletion of GFP-TDP1 (Fig.…”

Dynamic Sorting of Nuclear Components into Distinct Nucleolar Caps during Transcriptional Inhibition

“…These caps have been termed on occasion fibrillar caps as they contain FC proteins. These proteins include: RNA pol I, UBF, TBP, TAF I 63, and TAF I 110 (Reimer et al, 1987;Zatsepina et al, 1993;Jordan et al, 1996) and topoisomerase I (Christensen et al, 2004; Table 3). It has been shown that some nucleolar proteins can be found in both types of cap regions that contain DFC or FC proteins, such as Nopp140 (Chen et al, 1999) and MPP10.…”