Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint
Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint
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DOI：
10.1111/j.1356-9597.2004.00730.x
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发表时间：
2004-05-01
期刊：
影响因子：
2.1
通讯作者：
Toda, T
中科院分区：
文献类型：
--
作者：
Lehmann, A;Katayama, S;Toda, T
Skp1 是 E3 泛素连接酶 SCF (Skp1-Cullin-1-F-box) 的核心组件。它在 Cullin-1 和底物决定成分 F-box 蛋白之间形成适配器桥。为了确定 Skp1 的作用，使用诱变 PCR（聚合酶链式反应）进行温度敏感 (ts) 筛选，并分离出 9 个独立的 ts 突变体。将突变残基映射到人类 Skp1 的 3-D 结构上表明，突变体在与 F-box 蛋白的结合方面会受到影响，但与 Cullin-1 (Pcu1) 的结合不会受到影响。为了评估 ts Skp1 的结合特性，对 12 个 F-box 蛋白和 Pcu1 进行表位标记，并进行免疫共沉淀。该系统分析表明 ts Skp1 保留与 Pcu1 的结合。然而，与三种特定 F-box 蛋白（参与维持基因组完整性的必需 Pof1、Pof3 和非必需 Pof10）的结合减少。 skp1(ts) 细胞表现出 G2 细胞周期延迟，这归因于 DNA 损伤检查点的激活。有趣的是，与 pof3 突变体相反，在 pof3 突变体中，这个检查点是生存所必需的，skp1(ts) 中检查点的废除抑制了 G2 延迟，而且几乎挽救了 ts 表型。因此，pof3Delta 和 skp1(ts) 之间 DNA 损伤检查点的激活机制有所不同，这表明 Skp1 在检查点信号级联中具有新的作用。
Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1-F-box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1(ts) cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1(ts) suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3Delta and skp1(ts), implicating a novel role for Skp1 in the checkpoint-signalling cascade.