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

Abstract

DNA topoisomerase I releases torsion stress created by DNA transcription. In principle, this activity is required in the nucleoplasm for mRNA synthesis and in the nucleoli for rRNA synthesis. Yet, topoisomerase I is mostly a nucleolar protein. Current belief holds that this preference is triggered by the N-terminal domain of the enzyme, which constitutes a nucleolar import signal. Contradicting this view, we show here that nucleolar accumulation of various fragments of topoisomerase I is correlated with their lesser mobility in this compartment and not with the N-terminal domain being intact or present. Therefore, the N-terminal domain is not likely a nucleolar import signal. We show that it rather serves as an adaptor that anchors a subpopulation of topoisomerase I at fibrillar centers of nucleoli and nucleolar organizer regions of mitotic chromosomes. Thus, it provides a steady association of topoisomerase I with the rDNA and with RNA polymerase I, which is maintained in a living cell during the entire cell cycle.DNA topoisomerase I changes the pitch of DNA helices by cutting one DNA strand and allowing rotation of the other (1). One important role for this mechanism is the release of torsion stress created by DNA transcription. Thus, topoisomerase I activity should in principle be required in the nucleoplasm for the synthesis of mRNA, and in the nucleolus for the synthesis of rRNA. 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. In keeping with this, studies of genomic DNA cleavage by topoisomerase I have invariantly come up with sites in the rDNA (3-5). 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. We show here that in a living cell a sub population of topoisomerase I is constantly anchored at the fibrillar centers of the nucleolus or the nucleolar organizer region of mitotic chromosomes and that this is due to an adaptor function of the N-terminal domain of the enzyme. EXPERIMENTAL PROCEDURESCloning and Cell Culture-GFP 1 chimera of human topoisomerase I and of various fragments of it (see Fig. 3A) were stably expressed in the human embryonal kidney cell line 293 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) using the bicistronic expression vector pMC-2P (6) in which the translational initiation of the...

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

“…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 (TI ) was inserted into the vector pMC-EGFPP (17) by linker-PCR, giving rise to the construct TI -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.…”

“…Constructs and Cell Culture-Construction and characterization of cell lines supporting stable expression of GFP-topo I, GFP-topo I Phe-723 , GFP-topo I 190 -765 , and GFP-topo I has been documented in previous studies (5,12,15). Please note that in one of these studies (5) GFP-topo I has been incorrectly referred to as GFP-topo I 1-210 , leading to the misunderstanding that it has five residues less than it actually does.…”

“…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).…”

Phase 2 study of gemcitabine and irinotecan in metastatic breast cancer with correlatives to determine topoisomerase I localization as a predictor of response

“…8 Although this Nterminal domain does not contribute to the enzyme's catalytic activity in vitro, it is very important for in vivo activity, as the enzyme must enter the nucleus to exert its function. [9][10][11] Data have indicated that mutations within the N-terminus of top1 result in loss of nuclear localization and subsequent resistance to topoisomerase I inhibitors. 12 Localization of topo I can be determined using immunofluorescence microscopy in patients with hematologic and solid tumors.…”