Role for Nucleolin/Nsr1 in the Cellular Localization of Topoisomerase I

Abstract

Nucleolin functions in ribosome biogenesis and contains an acidic N terminus that binds nuclear localization sequences. In previous work we showed that human nucleolin associates with the N-terminal region of human topoisomerase I (Top1). We have now mapped the topoisomerase I interaction domain of nucleolin to the N-terminal 225 amino acids. We also show that the Saccharomyces cerevisiae nucleolin ortholog, Nsr1p, physically interacts with yeast topoisomerase I, yTop1p. Studies of isogenic NSR1 ؉ and ⌬nsr1 strains indicate that NSR1 is important in determining the cellular localization of yTop1p. Moreover, deletion of NSR1 reduces sensitivity to camptothecin, an antineoplastic topoisomerase I inhibitor. By contrast, ⌬nsr1 cells are hypersensitive to the topoisomerase II-targeting drug amsacrine. These findings indicate that nucleolin/Nsr1 is involved in the cellular localization of Top1 and that this localization may be important in determining sensitivity to drugs that target topoisomerases. Human Top11 is a nuclear protein involved in the regulation of DNA structure and is the target of an important new class of antineoplastic drugs, the camptothecins (1, 2). Studies in yeast provide convincing evidence that Top1 is the sole cellular target for camptothecins, with this knowledge facilitating attempts to understand the mechanisms by which the drug destroys cells (3-5). Top1 is a monomeric protein that relaxes supercoiled DNA by creating a transient single-strand nick, with this nick involving the covalent attachment of Top1 to the DNA phosphate backbone via a phosphotyrosine bond. Recent structural studies of Top1 and the Top1-DNA covalent complex led to molecular models of the mechanism by which camptothecins inhibit the catalytic activity of Top1 (6 -8). Nevertheless, formation of CPT-Top1-DNA ternary complexes is insufficient to explain the cytotoxic effects of CPT (9 -11). Current models invoke interactions between ternary complexes and replication or transcriptional machinery as being important in conversion of ternary complexes to lethal forms of DNA damage (12, 13). However, it is not known whether physical interactions between Top1 and other proteins (including proteins involved in replication or transcription) are important in the cytotoxic effects of CPT. Recently, an interaction between Top1 and the SV40 T antigen helicase was shown to modulate formation of Top1-CPT-DNA ternary complexes, suggesting that similar interactions between Top1 and cellular helicases may mediate the cytotoxicity of CPT (14). In addition, Top1 rapidly redistributes from the nucleolus to the nucleus or cytoplasm and is degraded after cellular exposure to . These alterations may confer transient cellular resistance to CPT and could be mediated by interactions between Top1 and other proteins.Top1 is known to physically interact with the following proteins: HMG17 (20), casein kinase II (21), RNA polymerase I (22), nucleolin (23), SV40 T antigen (24,25), p53 (26,27), the TATA-binding protein (28), the splicing factors SF2/ASF (2...

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

“…In fact, nucleolar accumulation of the active site mutant seemed to be precluded by its attenuated mobility in the nucleoplasm, rather than by alterations in the nucleolus. In summary, these data support our previous hypothesis that differences in protein mobility govern nucleolar localization of topoisomerase I (2), whereas our findings argue clearly against earlier suggestions that the capability of topoisomerase I accumulating inside the nucleoli should be a function of the N-terminal domain of the enzyme (8 -10, 12) or of parts of it (15). However, nucleolar accumulation due to a decreased mobility in this compartment seems be a feature not shared inadvertently by all proteins present in the nucleus because GFP alone exhibited a lesser intensity in the nucleoli (Fig.…”

“…2A) and thus seems to require cells to be alive. In contradiction to previous reports (15), the lack of co-localization with nucleolin shown in Fig. 3D (top) argues against a role of this protein in the nucleolar targeting of topoisomerase I.…”

“…Most notably, it seems to be a docking place for interacting proteins, such as nucleolin (27). Current belief holds that it also serves as a signal sequence triggering the nucleolar import of topoisomerase I (9,12), with nucleolin interactions possibly driving the process (15).…”

Interaction of Human Nuclear Topoisomerase I with Guanosine Quartet-forming and Guanosine-rich Single-stranded DNA and RNA Oligonucleotides

“…Top1 is known to play an important role in transcription (for review see Ref. 56) by direct binding to transcription complexes (57)(58)(59)(60) and phosphorylation of serinearginine rich (SR) splicing factors that control pre-mRNA splicing (61). Top1 enhances TFIID-TFIIA complex assembly during activation of transcription (62) and interacts with TFIIIC to promote accurate termination and reinitiation (63).…”

A Genetic Screen for Increased Loss of Heterozygosity inSaccharomyces cerevisiae

“…Like Lrs4 and Csm1, Top1 (topoisomerase I) is required for suppression of rDNA recombination (Christman et al 1988) and rDNA silencing (Smith et al 1999). While Top1 acts throughout the genome, it is highly enriched in the nucleolus where the rDNA resides (Edwards et al 2000). The nucleolar enrichment of Top1 is mediated in part by interactions with Tof2 (Park and Sternglanz 1999) and Fob1, both of which are localized to the rDNA and are required for Lrs4-and Csm1-mediated rDNA silencing .…”