Zhao Xiaolan, Rothstein Rodney
Department of Genetics and Development, Columbia University, College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032-2704, USA.
Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3746-51. doi: 10.1073/pnas.062502299.
Cell cycle checkpoints are evolutionarily conserved surveillance systems that protect genomic stability and prevent oncogenesis in mammals. One important target of checkpoint control is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP and DNA synthesis. In both yeast and humans, RNR is transcriptionally induced after DNA damage via Mec1/Rad53 (yeast) and ATM/CHK2 (human) checkpoint pathways. In addition, yeast checkpoint proteins Mec1 and Rad53 also regulate the RNR inhibitor Sml1. After DNA damage or at S phase, Mec1 and Rad53 control the phosphorylation and concomitant degradation of Sml1 protein. This new layer of control contributes to the increased dNTP production likely necessary for DNA repair and replication; however, the molecular mechanism is unclear. Here we show that Dun1, a downstream kinase of Mec1/Rad53, genetically and physically interacts with Sml1 in vivo. The absence of Dun1 activity leads to the accumulation of Sml1 protein at S phase and after DNA damage. As a result, dun1Delta strains need more time to finish DNA replication, are defective in mitochondrial DNA propagation, and are sensitive to DNA-damaging agents. Moreover, phospho-Sml1 is absent or dramatically reduced in dun1Delta cells. Finally, Dun1 can phosphorylate Sml1 in vitro. These results suggest that Dun1 kinase function is the last step required in the Mec1/Rad53 cascade to remove Sml1 during S phase and after DNA damage.
细胞周期检查点是进化上保守的监测系统，可保护哺乳动物的基因组稳定性并预防肿瘤发生。检查点控制的一个重要靶点是核糖核苷酸还原酶（RNR），它催化dNTP和DNA合成中的限速步骤。在酵母和人类中，DNA损伤后，RNR通过Mec1/Rad53（酵母）和ATM/CHK2（人类）检查点途径被转录诱导。此外，酵母检查点蛋白Mec1和Rad53还调节RNR抑制剂Sml1。DNA损伤后或在S期，Mec1和Rad53控制Sml1蛋白的磷酸化及随之而来的降解。这一新的控制层面有助于增加DNA修复和复制可能所需的dNTP产量；然而，分子机制尚不清楚。在这里，我们表明，Mec1/Rad53的下游激酶Dun1在体内与Sml1发生遗传和物理相互作用。Dun1活性的缺失导致Sml1蛋白在S期和DNA损伤后积累。结果，dun1Delta菌株需要更多时间来完成DNA复制，在线粒体DNA增殖方面存在缺陷，并且对DNA损伤剂敏感。此外，dun1Delta细胞中磷酸化的Sml1不存在或显著减少。最后，Dun1在体外可使Sml1磷酸化。这些结果表明，Dun1激酶功能是Mec1/Rad53级联反应在S期和DNA损伤后去除Sml1所需的最后一步。
