Ddb1 is required for the proteolysis of the Schizosaccharomyces pombe replication inhibitor Spd1 during S phase and after DNA damage.
Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression. A mutant S. pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype. We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1. However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear. It was shown that Ddb1 associates with the Cop9/signalosome. Moreover, the phenotypes of the Deltaddb1 strain are remarkably similar to the Deltacsn1 (or Deltacsn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome. Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase. Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. Here we show that a S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage. Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the Deltaddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Deltaddb1 strain. Our results establish an essential role of Ddb1 in the proteolysis of Spd1. In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome.
Bondar
Tanya
T
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Ponomarev
Aleksandr
A
Raychaudhuri
Pradip
P
D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002469:Cell Separation; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors; D016196:S Phase; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; C102445:Spd1 protein, S pombe; D025801:Ubiquitin; D004247:DNA; D012264:Ribonucleotide Reductases; D005982:Glutathione Transferase