title: Separation of replication and transcription domains in nucleoli.
abstract: In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: E
authors.initials: E
authors.lastname: Smirnov
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Borkovec
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: L
authors.initials: L
authors.lastname: Kováčik
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: S
authors.initials: S
authors.lastname: Svidenská
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: A
authors.initials: A
authors.lastname: Schröfel
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: M
authors.initials: M
authors.lastname: Skalníková
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: Z
authors.initials: Z
authors.lastname: Švindrych
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: P
authors.initials: P
authors.lastname: Křížek
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: M
authors.initials: M
authors.lastname: Ovesný
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: G M
authors.initials: GM
authors.lastname: Hagen
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: P
authors.initials: P
authors.lastname: Juda
authors.affiliation: Centre of Oncocytogenetics, Institute of Medical Biochemistry and Laboratory Diagnosis, General University Hospital and First Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: K
authors.initials: K
authors.lastname: Michalová
authors.affiliation: Department of Biology, Technische Universitat Darmstadt, Darmstadt, Germany.
authors.forename: M C
authors.initials: MC
authors.lastname: Cardoso
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: D
authors.initials: D
authors.lastname: Cmarko
authors.affiliation: Institute of Cell Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic.
authors.forename: I
authors.initials: I
authors.lastname: Raška
mesh_terms: D002460:Cell Line; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D014158:Transcription, Genetic
keywords: Nucleolus; Replication; Single-molecule localization microscopy; Super-resolution; Transcription; rDNA