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.

• MeSH
• buněčné jadérko genetika   metabolismus   MeSH
• buněčné linie MeSH
• genetická transkripce * MeSH
• HeLa buňky MeSH
• lidé MeSH
• replikace DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The essential role of MCM 2–7 proteins in the initiation of DNA replication in all eukaryotes is well known. Their role in replication elongation is supported by numerous studies, but there is still a knowledge gap in this respect. Even though biochemical studies have established an association of MCM proteins with replication forks, previous immunofluorescence studies in mammalian cells have suggested that MCM 2–7 proteins are displaced after replication initiation from sites of DNA replication. Therefore, we used a robust statistical method to more precisely analyse immunofluorescence localization of MCM 2 proteins with respect to the DNA replication foci. We show that despite the predominantly different localization of MCM 2 and replication signals, there is still a small but significant fraction of MCM 2 proteins that co-localize with DNA replication foci during most of S phase. The fluorescence localization of the MCM 2 proteins and DNA replication may thus reflect an active function of MCM 2 proteins associated with the replication foci and partially explain one facet of the “MCM paradox”.

• MeSH
• buněčné jádro metabolismus   MeSH
• chromatin metabolismus   MeSH
• financování organizované MeSH
• fluorescenční protilátková technika MeSH
• HeLa buňky MeSH
• jaderné proteiny metabolismus   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• neparametrická statistika MeSH
• počítačové zpracování obrazu metody   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• replikační počátek MeSH
• S fáze MeSH
• Check Tag
• lidé MeSH