Du Li-Lin, Nakamura Toru M, Moser Bettina A, Russell Paul
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Mol Cell Biol. 2003 Sep;23(17):6150-8. doi: 10.1128/MCB.23.17.6150-6158.2003.
The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.
裂殖酵母中的检查点蛋白Crb2与芽殖酵母中的Rad9以及人类的53BP1和BRCA1相关，有人提出它作为衔接蛋白，促进Rad3-Rad26激酶对特定底物的磷酸化。为了进一步了解其在检查点信号传导中的作用，我们通过荧光显微镜检查了其在活细胞中的定位。响应DNA损伤时，Crb2定位于不同的核灶，这些核灶代表DNA双链断裂（DSB）的位点。Crb2与Rad22在持续存在的灶中共定位，表明Crb2在修复过程中保留在DNA损伤位点。在Rad1、Rad3和Rad17复合物缺陷的细胞中，损伤诱导的Crb2灶仍然形成，但这些灶不会像在野生型细胞中那样持久存在。我们的结果表明，Crb2在DNA损伤位点发挥作用，其在损伤位点的受调控的持久定位可能参与促进DNA修复和/或在DNA修复进行时维持检查点停滞。
