Giant yeast cells with nonrecyclable ribonucleotide reductase
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. rnr1C883A rnr3Δ 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. rnr1C883A rnr3Δ 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. rnr1C883A rnr3Δ cells, which can be used as a protein overexpression system, thus illustrate the robustness of S. cerevisiae to multiple physiological parameters.
KeywordsRibonucleotide reductase Saccharomyces cerevisiae DNA replication DNA checkpoints Protein overexpression
We thank Jean Daraspe for expert technical assistance in electron microscopy, Jean-Yves Thuret for support in video-microscopy, Jean-Louis Sikorav for insightful comments, Sylvie Camier for discussions on RNR at the outset of this work, Etienne Schwob and Christelle de Renty for discussions on BrdU incorporation and detection and Sophie Chéruel for discussions on RNR constructs. This study was partly financed by grants from the Association pour la Recherche sur le Cancer (ARC) and from the Agence Nationale de la Recherche (ANR).
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