title: Spd2 assists Spd1 in the modulation of ribonucleotide reductase architecture but does not regulate deoxynucleotide pools.
abstract: In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The Schizosaccharomyces pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with sequence similarity to Spd1. We show that Spd2 is a CRL4(Cdt2)-controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3(ATR) checkpoint dependency of CRL4(Cdt2) mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4(Cdt2) mutant cells to the same extent as deletion of spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, whereas in combination with Spd2 it modulates RNR architecture and sensitizes cells to DNA damage.
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Vejrup-Hansen
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark NWCR Institute, School of Biological Sciences, Bangor University, Bangor, Gwynedd, LL57 2UW, UK.
authors.forename: Oliver
authors.initials: O
authors.lastname: Fleck
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark.
authors.forename: Katrine
authors.initials: K
authors.lastname: Landvad
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Fahnøe
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark.
authors.forename: Sebastian S
authors.initials: SS
authors.lastname: Broendum
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, UK.
authors.forename: Ann-Sofie
authors.initials: AS
authors.lastname: Schreurs
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark.
authors.forename: Birthe B
authors.initials: BB
authors.lastname: Kragelund
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 9RQ, UK.
authors.forename: Antony M
authors.initials: AM
authors.lastname: Carr
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Holmberg
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N., Denmark onigen@bio.ku.dk.
authors.forename: Olaf
authors.initials: O
authors.lastname: Nielsen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000494:Allosteric Regulation / Q000235:genetics; D000595:Amino Acid Sequence; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D064447:Checkpoint Kinase 2 / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D064267:Intrinsically Disordered Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009708:Nucleotidases / Q000378:metabolism; D011487:Protein Conformation; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C506543:Cdt2 protein, S pombe; D018797:Cell Cycle Proteins; D064267:Intrinsically Disordered Proteins; D029702:Schizosaccharomyces pombe Proteins; C102445:Spd1 protein, S pombe; C000597169:Spd2 protein, S pombe; D012264:Ribonucleotide Reductases; D064447:Checkpoint Kinase 2; C456840:rad3 protein, S pombe; D009708:Nucleotidases; C039170:deoxynucleotide 3'-phosphatase
keywords: CRL4Cdt2 ubiquitin ligase; Cancer model; Genome stability; Intrinsically disordered proteins; Ribonucleotide reductase; S. pombe