Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.

• MeSH
• Cell Nucleolus genetics   MeSH
• Epithelial Cells metabolism   MeSH
• Transcription, Genetic * MeSH
• HeLa Cells MeSH
• Kinetics MeSH
• Middle Aged MeSH
• Humans MeSH
• Limbus Corneae cytology   MeSH
• Cadaver MeSH
• Cold Temperature MeSH
• DNA, Ribosomal genetics   MeSH
• Ribosomes genetics   MeSH
• RNA, Ribosomal genetics   MeSH
• Aged MeSH
• Software MeSH
• Transfection MeSH
• Uridine analogs & derivatives   immunology   metabolism   MeSH
• Uridine Triphosphate analogs & derivatives   immunology   metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-bromouridine triphosphate MeSH Browser
• 5-fluorouridine MeSH Browser
• DNA, Ribosomal MeSH
• RNA, Ribosomal MeSH
• Uridine MeSH
• Uridine Triphosphate MeSH

DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.

• Keywords
• DNA repair, Lamins, Micro-irradiation, PCNA, S/G2 phases, rDNA,
• MeSH
• Cell Division genetics   physiology   MeSH
• Cell Nucleus metabolism   MeSH
• Cell Cycle genetics   physiology   MeSH
• Chromatin genetics   metabolism   MeSH
• G2 Phase genetics   physiology   MeSH
• DNA Repair genetics   physiology   MeSH
• Proliferating Cell Nuclear Antigen genetics   metabolism   MeSH
• S Phase genetics   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin MeSH
• Proliferating Cell Nuclear Antigen MeSH

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

• Keywords
• fibrillarin, fluctuation, pol I, rDNA, transcription pulsing,
• MeSH
• Cell Nucleolus chemistry   enzymology   MeSH
• Chromosomal Proteins, Non-Histone chemistry   metabolism   MeSH
• DNA-Directed RNA Polymerases chemistry   metabolism   MeSH
• HeLa Cells MeSH
• Immunohistochemistry MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• DNA-Directed RNA Polymerases MeSH
• fibrillarin MeSH Browser