Charton, Romain and Muguet, Alexia and Griesenbeck, Joachim and Smerdon, Michael J. and Conconi, Antonio (2019) In yeast cells arrested at the early S-phase by hydroxyurea, rRNA gene promoters and chromatin are poised for transcription while rRNA synthesis is compromised. MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS, 815. pp. 20-29. ISSN 0027-5107, 1873-135X
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Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase that is used as a chemotherapeutic agent to treat a number of chronic diseases. Addition of HU to cell cultures causes reduction of the dNTP cellular pool below levels that are required for DNA replication. This trigger dividing cells to arrest in early S-phase of the cell cycle. Cell division hinges on ribosome biogenesis, which is tightly regulated by rRNA synthesis. Remarkably, HU represses the expression of some genes the products of which are required for rRNA maturation. To gain more information on the cellular response to HU, we employed the yeast Saccharomyces cerevisiae as model organism and analyzed the changing aspects of closed to open forms of rRNA gene chromatin during cell cycle arrest, the arrangement of RNA polymerase-I (RNAPI) on the open genes, the presence of RNAPI transcription-factors, transcription and rRNA maturation. The rRNA gene chromatin structure was analyzed by psoralen crosslinking and the distribution of RNAPI was investigated by chromatin endogenous cleavage. In HU arrested cells nearly all rRNA genes were in the open form of chromatin, but only a portion of them was engaged with RNAPI. Analyses by chromatin immunoprecipitation confirmed that the overall formation of transcription pre-initiation complexes remained unchanged, suggesting that the onset of rRNA gene activation was not significantly affected by HU. Moreover, the in vitro transcription run-on assay indicated that RNAPI retained most of its transcription elongation activity. However, in HU treated cells, we found that (1) RNAPI accumulated next to the 5'-end of rRNA genes; (2) considerably less rRNA filaments were observed in electron micrographs of rDNA transcription units; and (3) rRNA maturation was compromised. It is established that HU inhibition of ribonucleotide reductase holds back DNA replication. This study indicates a hitherto unexplored cellular response to HU, namely altered rRNA synthesis, which could participate to hamper cell division.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | POLYMERASE-I; SACCHAROMYCES-CEREVISIAE; DNA-REPLICATION; RIBONUCLEOTIDE REDUCTASE; RDNA; REPAIR; FORK; ESTABLISHMENT; INHIBITION; NUCLEOSOME; Chromatin; Hydroxyurea; Chromatin endogenous cleavage; Psoralen crosslinking; rRNA genes; RNA polymerase I |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie III Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie III > Dr. Joachim Griesenbeck |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 09 Apr 2020 05:53 |
| Last Modified: | 09 Apr 2020 05:53 |
| URI: | https://pred.uni-regensburg.de/id/eprint/27048 |
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