Ndd1 Turnover by SCF^Grr1 Is Inhibited by the DNA Damage Checkpoint in <i>Saccharomyces cerevisiae</i>

In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage. Here, we show that Ndd1 is also subject to regulation by protein turnover during the mitotic cell cycle: Ndd1 is unstable during an unperturbed cell cycle, but is strongly stabilized in response to DNA damage. We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCFGrr1. In response to DNA damage, Ndd1 stabilization requires the checkpoint kinases Mec1/Tel1 and Swe1, the S. cerevisiae homolog of the Wee1 kinase. In both humans and yeast, the checkpoint promotes Wee1-dependent inhibitory phosphorylation of Cdk1 following exposure to DNA damage. While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood. We propose that the DNA damage checkpoint stabilizes Ndd1 by inhibiting Cdk1, which we show is required for targeting Ndd1 for destruction.在酿酒酵母（Saccharomyces cerevisiae）中，Ndd1是有丝分裂基因簇的专属转录激活因子，该基因簇包含33个编码关键有丝分裂调控因子的基因，因此Ndd1成为调控有丝分裂过程的核心枢纽。此前已有研究表明，包括细胞周期蛋白依赖性激酶（Cdk1）在内的多种激酶可通过磷酸化修饰Ndd1，以调控其在细胞周期中的活性。我们先前的研究证实，Ndd1会在DNA损伤响应中被磷酸化而受到抑制。本研究发现，Ndd1在有丝分裂细胞周期中还受到蛋白质周转的调控：在未受扰动的细胞周期内，Ndd1蛋白稳定性较差，但在DNA损伤响应条件下，其稳定性会被显著增强。我们的实验结果显示，Ndd1在有丝分裂中期的降解依赖于Cdk1活性与泛素连接酶SCFGrr1。当遭遇DNA损伤时，Ndd1的稳定则依赖于检查点激酶Mec1/Tel1以及Swe1——酿酒酵母中Wee1激酶的同源蛋白。无论是人类还是酿酒酵母，在暴露于DNA损伤后，检查点都会介导Wee1依赖的Cdk1抑制性磷酸化。尽管这一机制在多数生物中对检查点诱导的细胞周期阻滞至关重要，但在出芽酵母中并非如此，目前学界对损伤诱导的抑制性磷酸化在出芽酵母中的功能仍缺乏深入了解。我们据此提出：DNA损伤检查点通过抑制Cdk1来稳定Ndd1，而我们的研究证实Cdk1正是介导Ndd1被靶向降解的关键因子。