Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint
Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint
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DOI：
10.1128/mcb.00330-08
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发表时间：
2008-08-01
影响因子：
5.3
通讯作者：
Muzi-Falconi, Marco
中科院分区：
文献类型：
--
作者：
Puddu, Fabio;Granata, Magda;Muzi-Falconi, Marco
在基因毒性损伤后，真核细胞触发了一个被称为DNA损伤检查点反应的信号转导级联反应，其中包括将一个顶端激酶和几个下游因子装载到DNA上。染色质修饰在招募检查点蛋白中起重要作用。在出芽酵母中，甲基化的113-K79与检查点因子Rad9结合。Dot1的缺失阻止了113-K79甲基化，导致细胞周期G期的检查点缺陷和有丝分裂中检查点激活的减少，这表明另一种途径有助于M期Rad9的募集。我们发现复制因子Dpb11是第二种途径的关键。δ dpb11-1突变细胞对UV或Zeocin处理敏感，如果在M期照射，则不能激活Rad53。我们的数据表明，Dpb11通过与磷酸化的9-1-1复合物的相互作用保持在受损DNA附近，导致Rad9的模依赖性磷酸化。Dpb11在DNA损伤后也会磷酸化，而这种修饰在不可磷酸化的ddc1-T602A突变体中丢失。最后，我们表明，在体内，Dpb11与Dot1合作促进Rad9磷酸化，但也有助于Mec1激酶的充分激活。
Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated 113-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents 113-K79 methylation, leading to a checkpoint defect in the G, phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1 Delta dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mocldependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.