Suter Bernhard, Graham Christopher, Stagljar Igor
Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), Department of Biochemistry & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, ON, Canada.
Biotechniques. 2008 Nov;45(5):581-4. doi: 10.2144/000112949.
Two collections of chromosomally tagged yeast Saccharomyces cerevisiae strains were previously designed to detect protein-protein interactions via the Cross-and-Capture system. Here, we used these strain collections in a different application to screen for proteins that are phosphorylated in response to DNA damage by electrophoretic shift analysis. Modification of a number of proteins that are known targets for checkpoint kinases was confirmed this way. Furthermore, we identified the mismatch repair protein Pms1 as a novel target for phosphorylation in the response to DNA damage and replication fork arrest. Genetic analysis revealed that this phosphorylation is dependent on checkpoint activation by ATM and ATR (yeast Mec1p and Tel1p) kinase. Hence, we demonstrated that the Cross-and-Capture system could be efficiently used to detect posttranslational modifications that modulate and control protein function in eukaryotic cells.
之前设计了两组染色体标记的酿酒酵母菌株，用于通过交叉捕获系统检测蛋白质-蛋白质相互作用。在此，我们将这些菌株用于不同的应用，通过电泳迁移分析筛选响应DNA损伤而被磷酸化的蛋白质。通过这种方式证实了许多已知的检查点激酶靶蛋白的修饰。此外，我们鉴定出错配修复蛋白Pms1是响应DNA损伤和复制叉停滞时磷酸化的新靶点。遗传分析表明，这种磷酸化依赖于ATM和ATR（酵母Mec1p和Tel1p）激酶激活的检查点。因此，我们证明交叉捕获系统可有效地用于检测调节和控制真核细胞中蛋白质功能的翻译后修饰。
