Separation of replication and transcription domains in nucleoli
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
25450594
DOI
10.1016/j.jsb.2014.10.001
PII: S1047-8477(14)00210-X
Knihovny.cz E-resources
- Keywords
- Nucleolus, Replication, Single-molecule localization microscopy, Super-resolution, Transcription, rDNA,
- MeSH
- Cell Nucleolus genetics metabolism MeSH
- Cell Line MeSH
- Transcription, Genetic * MeSH
- HeLa Cells MeSH
- Humans MeSH
- DNA Replication * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
References provided by Crossref.org
Discontinuous transcription of ribosomal DNA in human cells
Fluctuations of pol I and fibrillarin contents of the nucleoli
Reproduction of the FC/DFC units in nucleoli
Nucleolar DNA: the host and the guests