title: Spd1 accumulation causes genome instability independently of ribonucleotide reductase activity but functions to protect the genome when deoxynucleotide pools are elevated.
abstract: Cullin4, Ddb1 and Cdt2 are core subunits of the ubiquitin ligase complex CRL4(Cdt2), which controls genome stability by targeting Spd1 for degradation during DNA replication and repair in fission yeast. Spd1 has an inhibitory effect on ribonucleotide reductase (RNR), the activity of which is required for deoxynucleotide (dNTP) synthesis. The failure to degrade Spd1 in mutants where CRL4(Cdt2) is defective leads to DNA integrity checkpoint activation and dependency. This correlates with a lower dNTP pool. Pools are restored in a spd1-deleted background and this also suppresses checkpoint activation and dependency. We hypothesized that fission yeast with RNR hyperactivity would display a mutator phenotype on their own, but also possibly repress aspects of the phenotype associated with the inability to target Spd1 for degradation. Here, we report that a mutation in the R1 subunit of ribonucleotide reductase cdc22 (cdc22-D57N), which alleviated allosteric feedback inhibition, caused a highly elevated dNTP pool that was further increased by deleting spd1. The Δspd1 cdc22-D57N double mutant had elevated mutation rates and was sensitive to damaging agents that cause DNA strand breaks, demonstrating that Spd1 can protect the genome when dNTP pools are high. In ddb1-deleted cells, cdc22-D57N also potently elevated RNR activity, but failed to allow cell growth independently of the intact checkpoint. Our results provide evidence that excess Spd1 interferes with other functions in addition to its inhibitory effect on ribonucleotide reduction to generate replication stress and genome instability.
authors.affiliation: Cell Cycle and Genome Stability Group, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark.
authors.forename: Oliver
authors.initials: O
authors.lastname: Fleck
authors.forename: Rasmus
authors.initials: R
authors.lastname: Vejrup-Hansen
authors.forename: Adam
authors.initials: A
authors.lastname: Watson
authors.forename: Antony M
authors.initials: AM
authors.lastname: Carr
authors.forename: Olaf
authors.initials: O
authors.lastname: Nielsen
authors.forename: Christian
authors.initials: C
authors.lastname: Holmberg
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D016681:Genome, Fungal; D042822:Genomic Instability; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C500125:Ddb1 protein, S pombe; D003854:Deoxyribonucleotides; D029702:Schizosaccharomyces pombe Proteins; C102445:Spd1 protein, S pombe; C080926:cdc22 protein, S pombe
keywords: Checkpoint kinase; Ddb1; Genome instability; Ribonucleotide reductase; Spd1