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

Mutat Res. 2019 May;815:20-29. doi: 10.1016/j.mrfmmm.2019.04.003. Epub 2019 Apr 27.

Abstract

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 immuno-precipitation 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.

Keywords: Chromatin; Chromatin endogenous cleavage; Hydroxyurea; Psoralen crosslinking; RNA polymerase I; rRNA genes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Cycle Checkpoints / drug effects*
  • Cell Cycle Checkpoints / genetics
  • Cell Division / genetics
  • Chromatin / genetics*
  • DNA Replication / genetics
  • DNA, Ribosomal / genetics
  • Genes, rRNA / genetics*
  • Hydroxyurea / pharmacology*
  • Promoter Regions, Genetic / genetics*
  • RNA Polymerase I / genetics
  • RNA, Ribosomal / genetics
  • S Phase / drug effects
  • S Phase / genetics
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / genetics
  • Transcription, Genetic / genetics*

Substances

  • Chromatin
  • DNA, Ribosomal
  • RNA, Ribosomal
  • Saccharomyces cerevisiae Proteins
  • RNA Polymerase I
  • Hydroxyurea