National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.
Nucleic Acids Res. 2012 Aug;40(15):7332-46. doi: 10.1093/nar/gks366. Epub 2012 May 8.
When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of Mcm10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that Mcm10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered Mcm10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates Mcm10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates Mcm10 in vitro, proving that Mcm10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets Mcm10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, Mcm10.
当哺乳动物细胞受到辐射损伤时，会停止 DNA 复制以防止错误的 DNA 合成。紫外线照射会触发 Mcm10 的蛋白水解，Mcm10 是一种必需的人类复制因子，会抑制正在进行的复制。在这里，我们报告 Mcm10 与包含 DNA 损伤结合蛋白 DDB1、cullin、Cul4 和环指蛋白 Roc1 的 E3 泛素连接酶相关联。DDB1、Roc1 或 Cul4 的消耗会消除 UV 触发的 Mcm10 蛋白水解，这意味着 Cul4-Roc1-DDB1 泛素连接酶介导 Mcm10 的下调。纯化的 Cul4-Roc1-DDB1 复合物在体外泛素化 Mcm10，证明 Mcm10 是其底物。通过筛选已知的 DDB1 相互作用蛋白，我们发现 VprBP 是将 Mcm10 作为降解目标的底物识别亚基。因此，这些结果表明 Cul4-DDB1-VprBP 泛素连接酶介导了应激诱导的复制因子 Mcm10 的蛋白水解。
