Molecular Genetics and Genomics

, Volume 285, Issue 5, pp 415–425 | Cite as

Giant yeast cells with nonrecyclable ribonucleotide reductase

  • Emilie Ma
  • Arach Goldar
  • Jean-Marc Verbavatz
  • Marie-Claude Marsolier-Kergoat
Original Paper

Abstract

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and thereby provides the precursors required for DNA synthesis and repair. In an attempt to test cell resistance to a permanent replicational stress, we constructed a mutant Saccharomyces cerevisiae strain containing exclusively nonrecyclable catalytic subunits of RNR that become inactivated following the reduction of one ribonucleoside diphosphate. In this rnr1C883A rnr3Δ mutant, the synthesis of each deoxyribonucleotide thus requires the production of one Rnr1C883A protein, which means that 26 million Rnr1C883A proteins (half the protein complement of a wild-type cell) have to be produced during each cell cycle. rnr1C883Arnr3Δ cells grow under constant replicational stress, as evidenced by the constitutive activation of the checkpoint effector Rad53, and their S phase is considerably extended compared to the wild type. rnr1C883Arnr3Δ mutants also display additional abnormalities such as a median cell volume increased by a factor of 8, and the presence of massive inclusion bodies. However, they exhibit a good plating efficiency and can be propagated indefinitely. rnr1C883Arnr3Δ cells, which can be used as a protein overexpression system, thus illustrate the robustness of S. cerevisiae to multiple physiological parameters.

Keywords

Ribonucleotide reductase Saccharomyces cerevisiae DNA replication DNA checkpoints Protein overexpression 

Supplementary material

438_2011_613_MOESM1_ESM.pdf (213 kb)
Supplementary material 1 (PDF 212 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Emilie Ma
    • 1
  • Arach Goldar
    • 1
  • Jean-Marc Verbavatz
    • 2
  • Marie-Claude Marsolier-Kergoat
    • 1
  1. 1.Institut de Biologie et de Technologies de Saclay, Service de Biologie Intégrative et de Génétique MoléculaireGif-sur-Yvette CedexFrance
  2. 2.Max Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany

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