DNA damoge checkpoint control in yeast

在真核生物中，ATM和ATR家族蛋白在DNA损伤和复制检查点控制中发挥着关键作用。这些蛋白的特征是具有与磷脂酰肌醇(PI)3-激酶相关的激酶结构域，但具有磷酸化蛋白质的能力。在发芽酵母中，ATR家族蛋白Mec1/ESR1对检查点反应和细胞生长是必不可少的。我们已经在两个杂交筛选中分离了PIE1基因，以寻找与Mec1相互作用的蛋白质，并表明PIE1在体内与Mec1物理相互作用。和Mec1一样，PIE1对细胞生长也是必不可少的，PIE1基因的缺失会导致DNA损伤和复制阻断检查点的缺陷，这与在Mec1突变体中观察到的情况类似。DNA损伤和复制阻断后的RAD53过度磷酸化在PIE1细胞中也减少，在Mec1细胞中发现。PIE1与裂解酵母Rad26有有限的同源性，后者与ATR家族蛋白Rad3形成复合体。PIE1中与Rad26同源的区域突变导致与PIE1突变类似的表型。Mec1蛋白激酶活性似乎对检查点反应和细胞生长至关重要。然而，Mec1激酶活性不受PIE1突变的影响，这表明PIE1除了调节Mec1激酶活性外，还调节其他一些重要功能。因此，PIE1在结构和功能上与Rad26相关，并与Mec1相互作用，控制检查点和细胞增殖。

In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol (PI) 3-kinase, but have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in atwo-hybrid screen for proteins that interact with Mec1 and show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1 is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1 mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1 cells as found in mec1 cells. Pie1 exhibits a limited homology to fission yeast Rad26 which forms a complex with the ATR family protein Rad3. Mutaion of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of the pie1 mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1 mutation, suggesting that Pie1 regulates some essential funtion other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26, and interacts with Mec1 to control checkpoints and cell proliferation.