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Post-translational modification of the ribonucleotide reductase inhibitor, Sml1, in response to DNA damage.

机译：响应DNA损伤，核糖核苷酸还原酶抑制剂Sml1的翻译后修饰。

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摘要

The production of dNTPs is necessary for DNA replication and genome maintenance in the face of genotoxic stress. Consequently, dNTP pools fluctuate throughout the cell cycle and increase following DNA damage treatment. Failure to properly regulate dNTP pools can lead to mutagenesis or cell death. Therefore, it is crucial that cells maintain appropriate levels of dNTPs throughout the cell cycle and in response to DNA damage. The concentration of dNTPs is largely controlled by precise regulation of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step in dNTP synthesis. The regulation of RNR is multifaceted and is largely controlled by the DNA damage checkpoint that includes the Mec1, Rad53 and Dun1 kinases. In Saccharomyces cerevisiae one level of RNR regulation is through a protein inhibitor Sml1. Sml1 binds to RNR and inhibits the activity of the enzyme. During S phase and following DNA damage treatment, Sml1 is phosphorylated by Dun1 and degraded. The studies described here identify two independent in vivo Dun1 phosphorylation events that are required for timely degradation of Sml1 following DNA damage treatment. We also show that Sml1 is ubiquitylated and its degradation depends on the 26S proteasome. Interestingly, YFP-Sml1, which usually localizes to the cytoplasm and is excluded from the nucleus, transiently relocalizes to the nucleus prior to degradation by the proteasome. Mutations that prevent the first phosphorylation event, sml1-SA1, completely block phosphorylation, nuclear localization and degradation of Sml1 following DNA damage treatment. On the other hand, blocking Sml1 phosphorylation, either by mutation (sml1-SA1) or by inhibiting the kinase (dun1Delta), does not inhibit Sml1 monoubiquitylation, indicating that Sml1 monoubiquitylation is independent of Dun1 phosphorylation. We propose a model in which Sml1 is monoubiquitylated and then phosphorylated by Dun1, causing it to dissociate from RNR and move into the nucleus. Nuclear-localized Sml1 is phosphorylated a second time by Dun1, which leads to its polyubiquitylation and targeting for degradation by the 26S proteasome. Study of the mechanism of Sml1 degradation, which is activated by the DNA damage checkpoint, provides insight into DNA damage-induced protein degradation. The complexity of regulation for this 104-amino acid Sml1 protein is surprising and underscores the importance of accurately maintaining appropriate levels of dNTP pools. Because Sml1 degradation is activated by the DNA damage checkpoint, these data provide insight into the mechanism of DNA damage-induced protein degradation.
机译：面对遗传毒性胁迫，dNTP的产生对于DNA复制和基因组维护是必需的。因此，dNTP池在整个细胞周期中波动，并在DNA损伤处理后增加。未能正确调节dNTP库可能导致诱变或细胞死亡。因此，至关重要的是，细胞在整个细胞周期中以及对DNA损伤的响应中都必须维持适当水平的dNTP。 dNTPs的浓度很大程度上受精确调节核糖核苷酸还原酶（RNR）的控制，该酶催化dNTP合成中的限速步骤。 RNR的调控是多方面的，并且在很大程度上受包括Mec1，Rad53和Dun1激酶在内的DNA损伤检查点的控制。在酿酒酵母中，RNR调节的一个水平是通过蛋白质抑制剂Sml1。 Sml1与RNR结合并抑制酶的活性。在S期和随后的DNA损伤处理期间，Sml1被Dun1磷酸化并降解。此处描述的研究确定了DNA损伤处理后Sml1的及时降解所需的两个独立的体内Dun1磷酸化事件。我们还显示Sml1被泛素化，其降解取决于26S蛋白酶体。有趣的是，YFP-Sml1通常位于细胞质中，不包含在细胞核中，在被蛋白酶体降解之前，它会短暂地重新定位于细胞核。防止第一个磷酸化事件的突变sml1-SA1完全阻断DNA损伤处理后Sml1的磷酸化，核定位和降解。另一方面，通过突变（sml1-SA1）或通过抑制激酶（dun1Delta）阻止Sml1磷酸化，不会抑制Sml1单泛素化，表明Sml1单泛素化与Dun1磷酸化无关。我们提出了一个模型，其中Sml1被泛素化然后被Dun1磷酸化，从而使其从RNR分离并进入细胞核。核定位的Sml1第二次被Dun1磷酸化，导致其多聚泛素化并靶向26S蛋白酶体降解。通过DNA损伤检查点激活的Sml1降解机理的研究，可以深入了解DNA损伤诱导的蛋白质降解。这种104个氨基酸的Sml1蛋白调节的复杂性令人惊讶，并强调了准确维持适当水平的dNTP库的重要性。因为Sml1降解是由DNA损伤检查点激活的，所以这些数据提供了对DNA损伤诱导的蛋白质降解机理的了解。

著录项

  • 作者

    Andreson, Bethany L.;

  • 作者单位

    Columbia University.;

  • 授予单位 Columbia University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 122 p.
  • 总页数 122
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:25

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