Spd 1 accumulation causes genome instability independently of ribonucleotide reduction but functions to protect the genome when deoxynucleotide pools are elevated

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. Failure to degrade Spd1 in CRL4 Cdt2 defective mutants leads to DNA integrity checkpoint activation and dependency. This correlates with reduced dNTP pools. Pools are restored in a spd1-deleted background and this also suppresses checkpoint activation and dependency. We hypothesized that fission yeast with RNR hyper activity would display a mutator phenotype on its own, but also possibly repress aspects of the phenotype associated with inability to target Spd1 for degradation. Here, we report that a mutation in the R1 subunit of ribonucleotide reductase cdc22 (cdc22-D57N) that alleviated allosteric feedback caused a highly elevated dNTP pool and that this 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 let them grow 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.