Bondar Tanya, Ponomarev Aleksandr, Raychaudhuri Pradip
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
J Biol Chem. 2004 Mar 12;279(11):9937-43. doi: 10.1074/jbc.M312570200. Epub 2003 Dec 29.
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.
最近我们发现，粟酒裂殖酵母的ddb1基因在S期进程中发挥作用。缺乏ddb1基因表达的粟酒裂殖酵母突变株在早期和晚期区域的复制都很缓慢，导致S期表型迟缓。我们将ddb1菌株中的表型归因于复制检查点激酶Cds1活性的增加。然而，ddb1菌株中Cds1高基础活性的原因尚不清楚。研究表明，Ddb1与Cop9信号体相关联。此外，Deltaddb1菌株的表型与缺乏Cop9信号体Csn1（或Csn2）亚基表达的Deltacsn1（或Deltacsn2）菌株非常相似。Cop9信号体与Pcu4协同作用，诱导Spd1的蛋白水解，Spd1通过抑制核糖核苷酸还原酶来抑制DNA复制。因此，我们研究了Spd1的蛋白水解是否需要Ddb1。在这里我们表明，缺乏Ddb1表达的粟酒裂殖酵母菌株在S期和DNA损伤后未能诱导Spd1的蛋白水解。此外，删除spd1基因会减弱缺乏ddb1表达的细胞中Cds1激酶的活性，这表明Spd1的积累导致了Deltaddb1菌株中Cds1活性的增加。此外，缺乏spd1和ddb1的双突变体不再表现出Deltaddb1菌株中观察到的生长缺陷和DNA损伤敏感性。我们的结果确定了Ddb1在Spd1蛋白水解中的重要作用。此外，该观察结果为Ddb1与Cop9信号体之间的功能联系提供了证据。
