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Deoxynucleoside salvage in fission yeast allows rescue of ribonucleotide reductase deficiency but not Spd1-Mediated inhibition of replication

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posted on 2023-06-09, 06:05 authored by Oliver Fleck, Ulrik Fahnøe, Katrine Vyff Løvschal, Marie-Fabrice Uwamahoro Gasasira, Irina N Marinova, Birthe B Kragelund, Antony CarrAntony Carr, Edgar Hartsuiker, Christian Holmberg, Olaf Nielsen
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.

Funding

Investigating the multiple mechanisms of RNR regulation by small disordered proteins; G0838; ASSOCIATION FOR INTERNATIONAL CANCER RESEARCH; 12-1118

History

Publication status

  • Published

File Version

  • Published version

Journal

Genes

ISSN

2073-4425

Publisher

Multidisciplinary Digital Publishing Institute

Issue

5

Volume

8

Article number

a128

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Research groups affiliated with

  • Genome Damage and Stability Centre Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2017-05-08

First Open Access (FOA) Date

2017-05-08

First Compliant Deposit (FCD) Date

2017-05-08

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