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Genes 2017, 8(5), 128; doi:10.3390/genes8050128

Deoxynucleoside Salvage in Fission Yeast Allows Rescue of Ribonucleotide Reductase Deficiency but Not Spd1-Mediated Inhibition of Replication

1
Cell Cycle and Genome Stability Group, Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark
2
North West Cancer Research Institute, Bangor University, Bangor, Gwynedd LL57 2UW, UK
3
Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark
4
Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK
Present address: Hvidovre Hospital and Department of International Health, Immunology and Microbiology, University of Copenhagen, DK-2650 Copenhagen, Denmark.
*
Author to whom correspondence should be addressed.
Academic Editor: Eishi Noguchi
Received: 9 March 2017 / Revised: 10 April 2017 / Accepted: 20 April 2017 / Published: 25 April 2017
(This article belongs to the Special Issue DNA Replication Controls)
View Full-Text   |   Download PDF [7350 KB, uploaded 28 April 2017]   |  

Abstract

In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA building blocks. The CRL4Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4Cdt2, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4Cdt2 inactivation, suggesting that Spd1—in addition to repressing dNTP synthesis—together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication. View Full-Text
Keywords: DNA replication; checkpoints; ribonucleotide reductase; PCNA; CRL4Cdt2; intrinsically disordered proteins; deoxynucleotide salvage; fission yeast DNA replication; checkpoints; ribonucleotide reductase; PCNA; CRL4Cdt2; intrinsically disordered proteins; deoxynucleotide salvage; fission yeast
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MDPI and ACS Style

Fleck, O.; Fahnøe, U.; Løvschal, K.V.; Gasasira, M.-F.U.; Marinova, I.N.; Kragelund, B.B.; Carr, A.M.; Hartsuiker, E.; Holmberg, C.; Nielsen, O. Deoxynucleoside Salvage in Fission Yeast Allows Rescue of Ribonucleotide Reductase Deficiency but Not Spd1-Mediated Inhibition of Replication. Genes 2017, 8, 128.

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