title: 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.
authors.affiliation: Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich Heine University, Medical School, Moorenstrasse 5, D-40225 Duesseldorf.
authors.forename: Morten O
authors.initials: MO
authors.lastname: Christensen
authors.forename: René M
authors.initials: RM
authors.lastname: Krokowski
authors.forename: Hans U
authors.initials: HU
authors.lastname: Barthelmes
authors.forename: Robert
authors.initials: R
authors.lastname: Hock
authors.forename: Fritz
authors.initials: F
authors.lastname: Boege
authors.forename: Christian
authors.initials: C
authors.lastname: Mielke
mesh_terms: D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D002466:Cell Nucleolus / Q000201:enzymology* / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004275:DNA, Ribosomal / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D017434:Protein Structure, Tertiary; D012318:RNA Polymerase I / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry; D013997:Time Factors; D059004:Topoisomerase I Inhibitors; D014158:Transcription, Genetic
chemical_list: D004275:DNA, Ribosomal; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D012335:RNA, Ribosomal; D059004:Topoisomerase I Inhibitors; D049452:Green Fluorescent Proteins; D003609:Dactinomycin; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I; D002166:Camptothecin