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Cullin4, Ddb1 and Cdt2 are core subunits of the ubiquitin ligase complex CRL4Cdt2, 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 CRL4Cdt2 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 Dspd1 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. [ABSTRACT FROM AUTHOR]

CDC25B is one of the three human phosphatases that activate the CDK-cyclin complexes, thereby triggering cell-cycle progression and division. Commitment to early mitotic events depends on the activation of a centrosomal pool of CDK1--cyclin-B1, and CDC25B is thought to be involved in initiating this centrosomal CDK1--cyclin-B1 activity. Centrosome-associated checkpoint kinase 1 (CHK1) has been proposed to contribute to the proper timing of a normal cell division cycle by inhibiting the activation of the centrosomal pool of CDK1. Here, we show that CDC25B is phosphorylated by CHK1 in vitro on multiple residues, including S230 and S563. We demonstrate these phosphorylations occur in vivo and that they are dependent on CHK1 activity. S230 CHK1-mediated phosphorylation is detected in cell extracts during S phase and G2 phase in the absence of DNA damage. We show that the S230-phosphorylated form of CDC25B is located at the centrosome from early S phase until mitosis. Furthermore, mutation of S230 to alanine increases the mitotic-inducing activity of CDC25B. Our results support a model in which, under normal cell cycle conditions and in the absence of DNA damage, CHK1 constitutively phosphorylates CDC25B during interphase and thus prevents the premature initiation of mitosis by negatively regulating the activity of CDC25B at the centrosome. [ABSTRACT FROM AUTHOR]